U.S. patent application number 17/237747 was filed with the patent office on 2021-10-28 for compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells.
The applicant listed for this patent is Inscripta, Inc.. Invention is credited to Phillip Belgrader, Jorge Bernate, Bruce Chabansky, Burak Dura, Christian Siltanen, Eric Smith, David Stumbo.
Application Number | 20210332388 17/237747 |
Document ID | / |
Family ID | 1000005651119 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210332388 |
Kind Code |
A1 |
Belgrader; Phillip ; et
al. |
October 28, 2021 |
COMPOSITIONS, METHODS, MODULES AND INSTRUMENTS FOR AUTOMATED
NUCLEIC ACID-GUIDED NUCLEASE EDITING IN MAMMALIAN CELLS
Abstract
This invention relates to compositions of matter, methods,
modules and instruments for automated mammalian cell growth,
reagent bundle creation and mammalian cell transfection followed by
nucleic acid-guided nuclease editing in live mammalian cells.
Inventors: |
Belgrader; Phillip;
(Pleasanton, CA) ; Siltanen; Christian; (Boulder,
CO) ; Dura; Burak; (Boulder, CO) ; Chabansky;
Bruce; (Boulder, CO) ; Stumbo; David;
(Boulder, CO) ; Smith; Eric; (Boulder, CO)
; Bernate; Jorge; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inscripta, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
1000005651119 |
Appl. No.: |
17/237747 |
Filed: |
April 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63092499 |
Oct 15, 2020 |
|
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|
63014944 |
Apr 24, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/04 20130101;
B01L 3/502761 20130101; B01L 2400/0424 20130101; C12N 15/11
20130101; B01L 2400/0421 20130101; C12N 9/22 20130101; C12N 15/907
20130101; B01L 7/00 20130101; C12N 2310/20 20170501; B01L 2300/161
20130101; B01L 2300/0681 20130101; B01L 2400/0415 20130101; C12M
23/12 20130101; C12N 2800/80 20130101; B01L 2300/123 20130101; C12M
23/50 20130101; C12M 23/16 20130101; C12N 15/1082 20130101; B01L
2200/0647 20130101 |
International
Class: |
C12N 15/90 20060101
C12N015/90; C12N 15/10 20060101 C12N015/10; C12M 3/06 20060101
C12M003/06; C12M 1/00 20060101 C12M001/00; C12M 1/32 20060101
C12M001/32; B01L 7/00 20060101 B01L007/00; C12N 15/11 20060101
C12N015/11; C12N 9/22 20060101 C12N009/22; B01L 3/00 20060101
B01L003/00 |
Claims
1. A method of transfecting and performing nucleic acid-guided
nuclease editing in mammalian cells in an automated editing
instrument comprising the steps of: providing an automated closed
cell editing instrument comprising a growth module and a
microfluidic module; growing mammalian cells in the growth module;
synthesizing a library of editing cassettes off-instrument, wherein
each editing cassette comprises a different gRNA and donor DNA
pair; generating, in the microfluidic module, a first plurality of
aqueous droplets in a first immiscible carrier fluid, wherein the
first plurality of aqueous droplets comprises dNTPs, primers,
polymerase and an editing cassette, and wherein each aqueous
droplet of the first plurality of aqueous droplets on average
comprises one or no editing cassette; providing, in the
microfluidic module, conditions to allow amplification of the
editing cassettes in the first plurality of aqueous droplets;
separating aqueous droplets with amplified editing cassettes from
aqueous droplets without amplified editing cassettes; generating,
in the microfluidic module, a second plurality of aqueous droplets
in a second immiscible carrier fluid, wherein the second plurality
of aqueous droplets comprises transfection reagents and a nucleic
acid-guided nuclease or nuclease fusion or a coding sequence for a
nucleic acid-guided nuclease or nuclease fusion; adding, in the
microfluidic module, the first plurality of aqueous droplets with
the amplified editing cassettes to the second immiscible carrier
fluid comprising the second plurality of aqueous droplets; merging,
in the microfluidic module, on average one of the first plurality
of aqueous droplets with the amplified editing cassette with on
average one of the second plurality of aqueous droplets comprising
transfection reagents resulting in aqueous droplet reagent bundles;
generating, in the microfluidic module, a third plurality of
aqueous droplets in a third immiscible carrier fluid, wherein the
third plurality of aqueous droplets comprises the mammalian cells
grown in the growth module; adding, in the microfluidic module, the
aqueous droplet reagent bundles to the third immiscible carrier
fluid comprising the third plurality of aqueous droplets comprising
the mammalian cells; merging, in the microfluidic module, on
average one aqueous droplet reagent bundle with on average one
aqueous droplet comprising the mammalian cells to produce merged
droplets; providing, in the microfluidic module, conditions for
cell transfection and editing; transferring the merged droplets to
the growth module; and demulsifying the merged droplets in the
growth module resulting in pooled merged droplets.
2. The method of claim 1, wherein the growth module is a rotating
growth module, a tangential flow filtration module or a bioreactor
module.
3. The method of claim 2, wherein the growth module is a rotating
growth module.
4. The method of claim 2, wherein the growth module is a tangential
flow filtration module.
5. The method of claim 2, wherein the growth module is a
bioreactor.
6. The method of claim 1, wherein the first, second and third
immiscible carrier fluids are the same immiscible carrier
fluid.
7. The method of claim 6, wherein the first, second and third
immiscible carrier fluids are fluorinated oils.
8. The method of claim 1, wherein at least two of the first, second
and third immiscible carrier fluids are different immiscible
carrier fluids.
9. The method of claim 1, wherein the mammalian cells are grown on
microcarriers.
10. The method of claim 1, wherein the merging step is accomplished
by a localized electric field, providing locally a chemical that
disrupts or destabilizes the surfactant in the second and/or third
immiscible carrier fluids, or use of a textured surface in a flow
path of a microfluidic channel through which the second and/or
third immiscible carrier fluids flow.
11. The method of claim 1, wherein the step of separating aqueous
droplets with amplified editing cassettes from aqueous droplets
without amplified editing cassettes is accomplished by
electrophoresis; dielectricphoresis; acoutstrophoresis; optical
sorting; or magnetophoresis.
12. The method of claim 11, wherein the step of separating aqueous
droplets with amplified editing cassettes from aqueous droplets
without amplified editing cassettes is accomplished by optical
sorting.
13. A method of transfecting and performing nucleic acid-guided
nuclease editing in mammalian cells in an automated editing
instrument comprising the steps of: providing an automated closed
cell editing instrument comprising a growth module, a microfluidic
module and a solid wall module; growing mammalian cells in the
growth module; synthesizing a library of editing cassettes
off-instrument, wherein each editing cassette comprises a different
gRNA and donor DNA pair; generating, in the microfluidic module, a
first plurality of aqueous droplets in a first immiscible carrier
fluid, wherein the first plurality of aqueous droplets comprise
dNTPs, primers, polymerase, a nucleic acid-guided nuclease or
nuclease fusion or a coding sequence for a nucleic acid-guided
nuclease or nuclease fusion and an editing cassette, wherein each
aqueous droplet of the first plurality of aqueous droplets on
average comprises one or no editing cassette; polymerizing, in the
microfluidic module, the first plurality of aqueous droplets
resulting in reagent bundle gel beads; providing, in the
microfluidic module, conditions to allow amplification of the
editing cassettes in the reagent bundle gel beads; separating
reagent bundle gel beads with amplified editing cassettes from
reagent bundle gel beads without amplified editing cassettes;
delivering the reagent bundle gel beads comprising amplified
editing cassettes to a solid wall module comprising wells, wherein
the wells comprise the mammalian cells and are sized so as to be
able to accommodate only one reagent bundle gel bead; dissolving,
in the solid wall module, the reagent bundle gel beads comprising
amplified editing cassettes in the wells; providing transfection
reagents to the wells in the solid wall module; providing
conditions to allow transfection and editing in the mammalian cells
in the solid wall module; growing the mammalian cells in the solid
wall module; dislodging the mammalian cells from the wells in the
solid wall module; and pooling the cells.
14. The method of claim 13, wherein the growth module is a rotating
growth module, a tangential flow filtration module or a bioreactor
module.
15. The method of claim 14, wherein the growth module is a rotating
growth module.
16. The method of claim 14, wherein the growth module is a
tangential flow filtration module.
17. The method of claim 14, wherein the growth module is a
bioreactor.
18. The method of claim 13, wherein the first, second and third
immiscible carrier fluids are the same immiscible carrier
fluid.
19. The method of claim 18, wherein the first, second and third
immiscible carrier fluids are fluorinated oils.
20. The method of claim 13, wherein at least two of the first,
second and third immiscible carrier fluids are different immiscible
carrier fluids.
21. The method of claim 13, wherein the mammalian cells are grown
on microcarriers.
22. The method of claim 13, wherein the step of separating aqueous
droplets with amplified editing cassettes from aqueous droplets
without amplified editing cassettes is accomplished by
electrophoresis; dielectricphoresis; acoutstrophoresis; optical
sorting; or magnetophoresis.
23. The method of claim 22, wherein the step of separating aqueous
droplets with amplified editing cassettes from aqueous droplets
without amplified editing cassettes is accomplished by optical
sorting.
24. The method of claim 13, wherein the solid wall module is a
solid wall isolation, incubation, and normalization (SWIIN)
module.
25. The method of claim 24, wherein the SWIIN module comprises
microwells with a volume of approximately 2.5 nl.
26. The method of claim 24, wherein the SWIIN module comprises
200,000 microwells.
27. The method of claim 24, wherein the SWIIN module comprises a
heater and a heated cover.
28. The method of claim 13, wherein the reagent bundle gel beads
comprise polyacrylamide with disulfide crosslinkers.
29. The method of claim 28, wherein the reagent bundle gel beads
are dissolved by exposure of the reagent bundle gel beads to a
reducing agent.
30. The method of claim 29, wherein the reducing agent is
.beta.-mercaptoethanol, dithiothreitol (DTT),
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), or
tris(2-carboxyethyl) phosphine (TCEP).
Description
RELATED CASES
[0001] This application claims priority to U.S. Ser. No. 63/092,499
filed 15 Oct. 2020, entitled "Compositions, Methods, Modules and
Instruments for Automated Nucleic Acid-Guided Nuclease Editing in
Mammalian Cells" and U.S. Ser. No. 63/014,944, filed 24 Apr. 2020,
also entitled "Compositions, Methods, Modules and Instruments for
Automated Nucleic Acid-Guided Nuclease Editing in Mammalian
Cells."
FIELD OF THE INVENTION
[0002] This invention relates to compositions of matter, methods,
modules and instruments for automated mammalian cell growth,
reagent bundle creation and mammalian cell transfection followed by
nucleic acid-guided nuclease editing in live mammalian cells.
BACKGROUND OF THE INVENTION
[0003] In the following discussion certain articles and methods
will be described for background and introductory purposes. Nothing
contained herein is to be construed as an "admission" of prior art.
Applicant expressly reserves the right to demonstrate, where
appropriate, that the methods referenced herein do not constitute
prior art under the applicable statutory provisions.
[0004] The ability to make precise, targeted changes to the genome
of living cells has been a long-standing goal in biomedical
research and development. Recently various nucleases have been
identified that allow manipulation of gene sequence; hence, gene
function. The nucleases include nucleic acid-guided nucleases,
which enable researchers to generate permanent edits in live cells.
Editing efficiencies frequently correlate with the concentration of
guide RNAs (gRNAs) and repair templates (e.g., donor DNAs homology
arms) in the cell, particularly in mammalian cells. That is, the
higher the concentration of gRNA and repair templates, the better
the editing efficiency. Further, it is desirable to be able to
perform many different edits in a population of mammalian cells
simultaneously and to do so in an automated fashion, minimizing
manual or hands-on cell manipulation.
[0005] There is thus a need in the art of nucleic acid-guided
nuclease gene editing for improved compositions, methods, modules
and instrumentation for increasing nucleic acid-guided nuclease
editing efficiency and throughput in live mammalian cells. The
present invention satisfies this need.
SUMMARY OF THE INVENTION
[0006] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key or essential features of the claimed subject matter, nor is it
intended to be used to limit the scope of the claimed subject
matter. Other features, details, utilities, and advantages of the
claimed subject matter will be apparent from the following written
Detailed Description including those aspects illustrated in the
accompanying drawings and defined in the appended claims.
[0007] The present disclosure relates to compositions, methods,
modules and automated instrumentation for making edits in a
mammalian genome. Efficient editing requires many excess copies of
editing cassettes--comprising a gRNA and a repair template (e.g.,
donor DNA)--in the cell nucleus. In order to perform
highly-multiplexed editing in a single reaction, it is necessary to
co-localize the cells with many clonal copies of each editing
cassette. Thus, the present compositions and methods entail making
"reagent bundles" comprising many (hundreds of thousands to
millions) clonal copies of an editing cassette and delivering or
co-localizing the reagent bundles with live mammalian cells such
that the editing cassettes edit the cells and the edited cells
continue to grow.
[0008] The automated instruments envisioned herein include a module
in which to grow the mammalian cells to be transfected, a module in
which to transfect and edit the cells, and a module in which to
pool and from which to collect the cells after editing. Herein are
described three alternative modules--a rotating growth module, a
tangential flow filtration module, and a bioreactor--for growing
the cells both on-instrument as one module of the automated
instrument or off-instrument prior to the start of the automated
process. Transfection and editing take place in a microfluidic
device or "chip" which are available commercially through, e.g.,
microfluidic ChipShop.TM. GmbH (Jena, Germany); uFluidix.TM.
(Toronto, Canada); Microflexis.TM. (Hamburg, Germany); and
microLIQUID.TM. (Gipuzkoa, Spain). Alternatively, once the cells
and reagents are encapsulated in droplets/emulsion, the
droplets/emulsion may be transferred or ported to the bioreactor
for incubation and editing, as well as subsequent emulsion
breaking. Moreover, the bioreactor may be used to generate the
emulsion using the impeller.
[0009] Thus, in some aspects there is provided a method for
transfecting and performing nucleic acid-guided nuclease editing in
mammalian cells in an automated editing instrument comprising the
steps of: providing an automated closed cell editing instrument
comprising a growth module and a microfluidic module; growing
mammalian cells in the growth module; synthesizing a library of
editing cassettes off-instrument, wherein each editing cassette
comprises a different gRNA and donor DNA pair; generating, in the
microfluidic module, a first plurality of aqueous droplets in a
first immiscible carrier fluid, wherein the first plurality of
aqueous droplets comprise dNTPs, primers, polymerase and an editing
cassette, and wherein each aqueous droplet of the first plurality
of aqueous droplets on average comprises one or no editing
cassette; providing, in the microfluidic module, conditions to
allow amplification of the editing cassettes in the first plurality
of aqueous droplets; separating aqueous droplets with amplified
editing cassettes from aqueous droplets without amplified editing
cassettes; generating, in the microfluidic module, a second
plurality of aqueous droplets in a second immiscible carrier fluid,
wherein the second plurality of aqueous droplets comprises
transfection reagents and a nucleic acid-guided nuclease or
nuclease fusion or a coding sequence for a nucleic acid-guided
nuclease or nuclease fusion; adding, in the microfluidic module,
the first plurality of aqueous droplets with the amplified editing
cassettes to the second immiscible carrier fluid comprising the
second plurality of aqueous droplets; merging, in the microfluidic
module, on average one of the first plurality of aqueous droplets
with the amplified editing cassette with on average one of the
second plurality of aqueous droplets comprising transfection
reagents resulting in aqueous droplet reagent bundles; generating,
in the microfluidic module, a third plurality of aqueous droplets
in a third immiscible carrier fluid, wherein the third plurality of
aqueous droplets comprises the mammalian cells grown in the growth
module; adding, in the microfluidic module, the aqueous droplet
reagent bundles to the third immiscible carrier fluid comprising
the third plurality of aqueous droplets comprising the mammalian
cells; merging, in the microfluidic module, on average one aqueous
droplet reagent bundle with on average one aqueous droplet
comprising the mammalian cells to produce merged droplets;
providing, in the microfluidic module, conditions for cell
transfection and editing; transferring the merged droplets to the
growth module; and demulsifying the merged droplets in the growth
module resulting in pooled merged droplets.
[0010] In some aspects of this method, the merging step is
accomplished by a localized electric field, providing locally a
chemical that disrupts or destabilizes the surfactant in the second
and/or third immiscible carrier fluids, or use of a textured
surface in a flow path of a microfluidic channel through which the
second and/or third immiscible carrier fluids flow. In some
embodiments, the first, second and third immiscible carrier fluids
are the same immiscible carrier fluid, and in some aspects, the
first, second and third immiscible carrier fluids are a different
immiscible carrier fluid.
[0011] Also provided herein is a method of transfecting and
performing nucleic acid-guided nuclease editing in mammalian cells
in an automated editing instrument comprising the steps of:
providing an automated closed cell editing instrument comprising a
growth module, a microfluidic module and a solid wall module;
growing mammalian cells in the growth module; synthesizing a
library of editing cassettes off-instrument, wherein each editing
cassette comprises a different gRNA and donor DNA pair; generating,
in the microfluidic module, a first plurality of aqueous droplets
in a first immiscible carrier fluid, wherein the first plurality of
aqueous droplets comprise dNTPs, primers, polymerase, a nucleic
acid-guided nuclease or nuclease fusion or a coding sequence for a
nucleic acid-guided nuclease or nuclease fusion and an editing
cassette, wherein each aqueous droplet of the first plurality of
aqueous droplets on average comprises one or no editing cassette;
polymerizing, in the microfluidic module, the first plurality of
aqueous droplets resulting in reagent bundle gel beads; providing,
in the microfluidic module, conditions to allow amplification of
the editing cassettes in the reagent bundle gel beads; separating
reagent bundle gel beads with amplified editing cassettes from
reagent bundle gel beads without amplified editing cassettes;
delivering the reagent bundle gel beads comprising amplified
editing cassettes to a solid wall module comprising wells, wherein
the wells comprise the mammalian cells and are sized so as to be
able to accommodate only one reagent bundle gel bead; dissolving,
in the solid wall module, the reagent bundle gel beads comprising
amplified editing cassettes in the wells; providing transfection
reagents to the wells in the solid wall module; providing
conditions to allow transfection and editing in the mammalian cells
in the solid wall module; growing the mammalian cells in the solid
wall module; dislodging the mammalian cells from the wells in the
solid wall module; and pooling the cells.
[0012] In some aspects of this embodiment, the solid wall module is
a solid wall isolation, incubation, and normalization (SWIIN)
module, and in some aspects, the SWIIN module comprises at least
200,000 microwells with a volume of approximately 2.5 nl. In some
aspects, the SWIIN module comprises a heater and a heated
cover.
[0013] Also in some aspects of this embodiment, the reagent bundle
gel beads comprise polyacrylamide with disulfide crosslinkers, and
in some aspects, the reagent bundle gel beads are dissolved by
exposure of the reagent bundle gel beads to a reducing agent such
as .beta.-mercaptoethanol, dithiothreitol (DTT),
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), or
tris(2-carboxyethyl) phosphine (TCEP). In some embodiments, the
first, second and third immiscible carrier fluids are the same
immiscible carrier fluid, and in some aspects, the first, second
and third immiscible carrier fluids are a different immiscible
carrier fluid.
[0014] In yet another embodiment, there is provided a method of
transfecting and performing nucleic acid-guided nuclease editing in
mammalian cells in an automated editing instrument comprising the
steps of: providing an automated closed cell editing instrument
comprising a growth module, a microfluidic module and a solid wall
module; growing mammalian cells in the growth module; synthesizing
a library of editing cassettes off-instrument, wherein each editing
cassette comprises a different gRNA and donor DNA pair; generating,
in the microfluidic module, a first plurality of aqueous droplets
in a first immiscible carrier fluid, wherein the first plurality of
aqueous droplets comprise dNTPs, primers, polymerase, a nucleic
acid-guided nuclease or nuclease fusion or a coding sequence for a
nucleic acid-guided nuclease or nuclease fusion and an editing
vector, wherein each aqueous droplet in the first plurality of
aqueous droplets on average comprises one or no editing vector and
wherein each aqueous droplet also comprises a transformation agent;
polymerizing, in the microfluidic module, the first plurality of
aqueous droplets resulting in reagent bundle gel beads, wherein the
reagent bundle gel beads are degradable; providing, in the
microfluidic module, conditions to allow amplification of the
editing cassettes in the reagent bundle gel beads; functionalizing
a surface of the polymerized reagent bundle gel beads, wherein the
functionalizing allows for capture of mammalian cells on the
surface of the polymerized reagent bundle gel beads; depositing the
mammalian cells grown in the growth module on the surface of the
polymerized reagent bundle gel beads; co-localizing, in an editing
module, non-degradable microcarriers with a functionalized surface
with the functionalized polymerized reagent bundle gel beads,
wherein the functionalized surface on the non-degradable
micorcarriers allows for capture of mammalian cells on the surface
of the non-degradable microcarriers; degrading, in an editing
module, the polymerized reagent bundle gel beads to release the
editing vectors and to release the mammalian cells from the surface
of the polymerized reagent bundle gel beads; and capturing the
mammalian cells on the surface of the non-degradable
microcarriers.
[0015] In aspects of all these embodiments, the growth module may
be a rotating growth module, a tangential flow filtration module or
a bioreactor module. In some aspects of these embodiments, the
mammalian cells are grown on microcarriers.
[0016] In some aspects of these embodiments, the step of sorting
cells, droplets or gel beads is accomplished by electrophoresis;
dielectricphoresis; acoutstrophoresis; optical sorting; or
magnetophoresis.
[0017] These aspects and other features and advantages of the
invention are described below in more detail.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
[0019] FIG. 1A depicts an exemplary workflow employing
liquid-partitioned (e.g., droplet) delivery for editing mammalian
cells grown in suspension. FIG. 1B depicts one embodiment of a
droplet generator used in this FIG. 1B to partition editing
cassettes in a Poisson distribution. FIG. 1C depicts an exemplary
method for generating partitioned clonal copies of editing
cassettes and transfecting and editing live mammalian cells in the
droplet partitions.
[0020] FIG. 2A depicts an exemplary workflow employing solid wall
partitioning and editing of mammalian cells grown in suspension.
FIG. 2B depicts an exemplary method for encapsulating clonal copies
of editing cassettes in gel beads. FIG. 2C depicts an embodiment of
a method for preparing gel bead reagent bundles for transfecting
cells in a solid wall partitioning module. FIG. 2D depicts
transfecting and editing live mammalian cells in a solid wall
partitioning module.
[0021] FIG. 3A depicts one embodiment of a growth module,
consisting of a rotating growth vial for use with the cell growth
module described herein and in relation to FIGS. 3C-3E. FIG. 3B
illustrates a top-down view of the rotating growth vial depicted in
FIG. 3A, showing optional internal "fins" or "paddles" for growing
mammalian cells. FIG. 3C is a perspective view of one embodiment of
a rotating growth vial in a cell growth module housing. FIG. 3D
depicts a cut-away view of the cell growth module from FIG. 3C.
FIG. 3E illustrates the cell growth module of FIG. 3C coupled to
LED, detector, and temperature regulating components.
[0022] FIG. 4A depicts retentate (top) and permeate (middle)
members for use in a tangential flow filtration module (e.g., cell
growth and/or concentration module), as well as the retentate and
permeate members assembled into a tangential flow assembly
(bottom). FIG. 4B depicts two side perspective views of a reservoir
assembly of a tangential flow filtration module. FIGS. 4C-4E depict
an exemplary top, with fluidic and pneumatic ports and gasket
suitable for the reservoir assemblies shown in FIG. 4B. FIGS. 4F
and 4G depict a retentate reservoir assembly comprising one or more
strainers or sieves which may be used to dissociate cells in the
tangential flow filtration module.
[0023] FIGS. 5A-5G depict various components of an embodiment of a
bioreactor useful for growing and transducing mammalian cells by
the methods described herein. FIG. 5H-1 and FIG. 5H-2 depicts an
exemplary fluidic diagram for the bioreactor described in relation
to FIGS. 5A-5G. FIG. 5I depicts an exemplary control system block
diagram for the bioreactor described in relation to FIGS.
5A-5G.
[0024] FIGS. 6A-6C depict an embodiment of a solid wall isolation
incubation and normalization (SWIIN) module. FIG. 6D depicts the
embodiment of the SWIIN module in FIGS. 6A-6C further comprising a
heater and a heated cover.
[0025] FIG. 7A is a representation of a photomicrograph of aqueous
droplets comprising a lipoplex assembly in an immiscible carrier
fluid and a representation of a photomicrograph of the demulsified
lipoplex assembly being combined with mammalian cells in aqueous
droplets in an immiscible carrier fluid. FIG. 7B is a bar graph of
the efficiency of HEK293T cells transfected in bulk and HEK293T
cells transfected in droplets where the droplets are not washed or
are washed post-transfection.
[0026] FIG. 8A depicts a method for determining purity of cells
after transfection and demulsification. FIG. 8B is a bar graph
showing the retained purity of cells transfected, edited and
demulsified by the methods described herein.
[0027] FIG. 9A is a graph plotting the copy number of
differently-sized amplified nucleic acids (e.g., 500 bp, 2000 bp
and 10,000 bp) per droplet in differently-sized droplets. FIG. 9B
is a graph plotting the cell coverage for 500 bp amplicons for
droplets of differing size.
[0028] FIG. 10 depicts an engine plasmid and a library of amplified
editing cassettes.
[0029] FIG. 11 depicts a map for an editing cassette.
[0030] FIGS. 12A and 12B are graphs demonstrating that the
materials comprising the components of the bioreactor are
biocompatible.
[0031] FIG. 13 comprises three graphs demonstrating that iPSC
culture and cell expansion in the bioreactor described herein is
comparable to cell culture and expansion in a CORNING.RTM. spinner
flask and in a traditional cell culture plate.
[0032] FIG. 14 is a graph showing that media exchange at .about.200
ml/minute does not impact cell growth.
[0033] FIG. 15 is a series of four graphs demonstrating that up to
five rounds of impeller shear is tolerated by iPSCs with no
negative effects on re-seeding.
[0034] FIG. 16 shows a workflow at upper right, a table reporting
percent efficiency at various steps in the workflow at lower right,
and a graph showing the replicates measuring the percent efficiency
at various steps in the workflow at left.
[0035] FIG. 17 is a graph showing that cell seeding and expansion
are both unaffected by the impeller-shear based passaging
protocol.
[0036] FIG. 18 at top are histograms showing the fluorescent
expression distribution measured via flow cytometry of the cell
population for individual stemness marker expression. FIG. 18 at
bottom left is a bar graph showing a stemness panel (FACS %
positive) for cells in the bioreactor described herein, on laminin
plates and on MATRIGEL.RTM. plates (CORNING.RTM. BIOCOAT.TM.
MATRIGEL.RTM. 6-well plates (Corning, Inc., Glendale, Ariz.)). FIG.
18 at bottom right is a bar graph showing a stemness panel (FACS
median fluorescence) for cells in the bioreactor described herein,
on laminin plates and on MATRIGEL.RTM. plates (CORNING.RTM.
BIOCOAT.TM. MATRIGEL.RTM. 6-well plates (Corning, Inc., Glendale,
Ariz.)).
[0037] FIG. 19A-19F show a series of panels, both % positive and
median fluorescence, demonstrating that iPSCs grown in the
bioreactor described herein maintain differentiation potential
comparable to iPSCs cultured on laminin plates and in MATRIGEL.RTM.
plates (CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates
(Corning, Inc., Glendale, Ariz.)).
[0038] It should be understood that the drawings are not
necessarily to scale, and that like reference numbers refer to like
features.
DETAILED DESCRIPTION
[0039] All of the functionalities described in connection with one
embodiment are intended to be applicable to the additional
embodiments described herein except where expressly stated or where
the feature or function is incompatible with the additional
embodiments. For example, where a given feature or function is
expressly described in connection with one embodiment but not
expressly mentioned in connection with an alternative embodiment,
it should be understood that the feature or function may be
deployed, utilized, or implemented in connection with the
alternative embodiment unless the feature or function is
incompatible with the alternative embodiment.
[0040] The practice of the techniques described herein may employ,
unless otherwise indicated, conventional techniques and
descriptions of organic chemistry, polymer technology, molecular
biology (including recombinant techniques), cell biology,
biochemistry and sequencing technology, which are within the skill
of those who practice in the art. Such conventional techniques
include polymer array synthesis, hybridization and ligation of
polynucleotides, and detection of hybridization using a label.
Specific illustrations of suitable techniques can be had by
reference to the examples herein. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Green, et al., Eds. (1999), Genome
Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel,
Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual;
Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory
Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis;
Sambrook and Russell (2006), Condensed Protocols from Molecular
Cloning: A Laboratory Manual; and Sambrook and Russell (2002),
Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.
Freeman, New York N.Y.; Gait, "Oligonucleotide Synthesis: A
Practical Approach" 1984, IRL Press, London; Nelson and Cox (2000),
Lehninger, Principles of Biochemistry 3.sup.rd Ed., W. H. Freeman
Pub., New York, N.Y.; Viral Vectors (Kaplift & Loewy, eds.,
Academic Press 1995); all of which are herein incorporated in their
entirety by reference for all purposes. For mammalian/stem cell
culture and methods see, e.g., Basic Cell Culture Protocols, Fourth
Ed. (Helgason & Miller, eds., Humana Press 2005); Culture of
Animal Cells, Seventh Ed. (Freshney, ed., Humana Press 2016);
Microfluidic Cell Culture, Second Ed. (Borenstein, Vandon, Tao
& Charest, eds., Elsevier Press 2018); Human Cell Culture
(Hughes, ed., Humana Press 2011); 3D Cell Culture (Koledova, ed.,
Humana Press 2017); Cell and Tissue Culture: Laboratory Procedures
in Biotechnology (Doyle & Griffiths, eds., John Wiley &
Sons 1998); Essential Stem Cell Methods, (Lanza & Klimanskaya,
eds., Academic Press 2011); Stem Cell Therapies: Opportunities for
Ensuring the Quality and Safety of Clinical Offerings: Summary of a
Joint Workshop (Board on Health Sciences Policy, National Academies
Press 2014); Essentials of Stem Cell Biology, Third Ed., (Lanza
& Atala, eds., Academic Press 2013); and Handbook of Stem
Cells, (Atala & Lanza, eds., Academic Press 2012). For
background on microfluidic-based emulsion formation and reactions,
see Niu and deMello, "Building droplet-based microfluidic systems
for biological analysis", Biochem Soc. Trans., 40:615-623 (2012);
Solvas and deMello, "Droplet microfluidics: recent developments and
future applications", Chem. Commun., 47:1936-42 (2011); and
Macosko, et al., Cell, 161:1202-14 (2015). CRISPR-specific
techniques can be found in, e.g., Genome Editing and Engineering
from TALENs and CRISPRs to Molecular Surgery, Appasani and Church
(2018); and CRISPR: Methods and Protocols, Lindgren and Charpentier
(2015); which is incorporated herein in its entirety by reference
for all purposes.
[0041] Note that as used herein and in the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an oligonucleotide" refers to one or more
oligonucleotides, and reference to "an automated system" includes
reference to equivalent steps and methods for use with the system
known to those skilled in the art, and so forth. Additionally, it
is to be understood that terms such as "left," "right," "top,"
"bottom," "front," "rear," "side," "height," "length," "width,"
"upper," "lower," "interior," "exterior," "inner," "outer" that may
be used herein merely describe points of reference and do not
necessarily limit embodiments of the present disclosure to any
particular orientation or configuration. Furthermore, terms such as
"first," "second," "third," etc., merely identify one of a number
of portions, components, steps, operations, functions, and/or
points of reference as disclosed herein, and likewise do not
necessarily limit embodiments of the present disclosure to any
particular configuration or orientation.
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications mentioned herein are incorporated by reference for the
purpose of describing and disclosing devices, methods and cell
populations that may be used in connection with the presently
described invention.
[0043] Where a range of values is provided, it is understood that
each intervening value, between the upper and lower limit of that
range and any other stated or intervening value in that stated
range is encompassed within the invention. The upper and lower
limits of these smaller ranges may independently be included in the
smaller ranges, and are also encompassed within the invention,
subject to any specifically excluded limit in the stated range.
Where the stated range includes one or both of the limits, ranges
excluding either or both of those included limits are also included
in the invention.
[0044] In the following description, numerous specific details are
set forth to provide a more thorough understanding of the present
invention. However, it will be apparent to one of ordinary skill in
the art that the present invention may be practiced without one or
more of these specific details. In other instances, features and
procedures well known to those skilled in the art have not been
described in order to avoid obscuring the invention.
[0045] The term "complementary" as used herein refers to
Watson-Crick base pairing between nucleotides and specifically
refers to nucleotides hydrogen bonded to one another with thymine
or uracil residues linked to adenine residues by two hydrogen bonds
and cytosine and guanine residues linked by three hydrogen bonds.
In general, a nucleic acid includes a nucleotide sequence described
as having a "percent complementarity" or "percent homology" to a
specified second nucleotide sequence. For example, a nucleotide
sequence may have 80%, 90%, or 100% complementarity to a specified
second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10
of 10 nucleotides of a sequence are complementary to the specified
second nucleotide sequence. For instance, the nucleotide sequence
3'-TCGA-5' is 100% complementary to the nucleotide sequence
5'-AGCT-3'; and the nucleotide sequence 3'-TCGA-5' is 100%
complementary to a region of the nucleotide sequence
5'-TAGCTG-3'.
[0046] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites, nuclear localization
sequences, enhancers, and the like, which collectively provide for
the replication, transcription and translation of a coding sequence
in a recipient cell. Not all of these types of control sequences
need to be present so long as a selected coding sequence is capable
of being replicated, transcribed and--for some
components--translated in an appropriate host cell.
[0047] As used herein the term "donor DNA" or "donor nucleic acid"
or "homology arm" or "repair arm" refers to nucleic acid that is
designed to introduce a DNA sequence modification (insertion,
deletion, substitution) into a locus by homologous recombination
using nucleic acid-guided nucleases or a nucleic acid that serves
as a template (including a desired edit) to be incorporated into
target DNA by reverse transcriptase in a CREATE fusion system. For
homology-directed repair, the donor DNA must have sufficient
homology to the regions flanking the "cut site" or the site to be
edited in the genomic target sequence. The length of the homology
arm(s) will depend on, e.g., the type and size of the modification
being made. In many instances and preferably, the donor DNA will
have two regions of sequence homology (e.g., two homology arms) to
the genomic target locus. Preferably, an "insert" region or "DNA
sequence modification" region--the nucleic acid modification that
one desires to be introduced into a genome target locus in a
cell-will be located between two regions of homology. The DNA
sequence modification may change one or more bases of the target
genomic DNA sequence at one specific site or multiple specific
sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base
pairs of the target sequence. A deletion or insertion may be a
deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50,
75, 100, 150, 200, 300, 400, or 500 or more base pairs of the
target sequence.
[0048] The terms "editing cassette", "CREATE cassette", "CREATE
editing cassette", or "CREATE fusion cassette" refers to a nucleic
acid molecule comprising a coding sequence for transcription of a
guide nucleic acid or gRNA covalently linked to a coding sequence
for transcription of a donor DNA or homology arm.
[0049] The terms "guide nucleic acid" or "guide RNA" or "gRNA"
refer to a polynucleotide comprising 1) a guide sequence capable of
hybridizing to a genomic target locus, and 2) a scaffold sequence
capable of interacting or complexing with a nucleic acid-guided
nuclease.
[0050] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or, more often in the context of
the present disclosure, between two nucleic acid molecules. The
term "homologous region" or "homology arm" refers to a region on
the donor DNA with a certain degree of homology with the target
genomic DNA sequence. Homology can be determined by comparing a
position in each sequence which may be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same base or amino acid, then the molecules are homologous at
that position. A degree of homology between sequences is a function
of the number of matching or homologous positions shared by the
sequences.
[0051] "Nucleic acid-guided editing components" refers to one,
some, or all of a nucleic acid-guided nuclease or nickase fusion
enzyme, a guide nucleic acid and a donor nucleic acid.
[0052] "Operably linked" refers to an arrangement of elements where
the components so described are configured so as to perform their
usual function. Thus, control sequences operably linked to a coding
sequence are capable of effecting the transcription, and in some
cases, the translation, of a coding sequence. The control sequences
need not be contiguous with the coding sequence so long as they
function to direct the expression of the coding sequence. Thus, for
example, intervening untranslated yet transcribed sequences can be
present between a promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence. In fact, such sequences need not reside on the
same contiguous DNA molecule (i.e., chromosome) and may still have
interactions resulting in altered regulation.
[0053] A "PAM mutation" refers to one or more edits to a target
sequence that removes, mutates, or otherwise renders inactive a PAM
or spacer region in the target sequence.
[0054] A "promoter" or "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase and initiating
transcription of a polynucleotide or polypeptide coding sequence
such as messenger RNA, ribosomal RNA, small nuclear or nucleolar
RNA, guide RNA, or any kind of RNA. Promoters may be constitutive
or inducible.
[0055] As used herein the term "selectable marker" refers to a gene
introduced into a cell, which confers a trait suitable for
artificial selection. General use selectable markers are well-known
to those of ordinary skill in the art. Drug selectable markers such
as ampicillin/carbenicillin, kanamycin, chloramphenicol,
nourseothricin N-acetyl transferase, erythromycin, tetracycline,
gentamicin, bleomycin, streptomycin, puromycin, hygromycin,
blasticidin, and G418 may be employed. In other embodiments,
selectable markers include, but are not limited to human nerve
growth factor receptor (detected with a MAb, such as described in
U.S. Pat. No. 6,365,373); truncated human growth factor receptor
(detected with MAb); mutant human dihydrofolate reductase (DHFR;
fluorescent MTX substrate available); secreted alkaline phosphatase
(SEAP; fluorescent substrate available); human thymidylate synthase
(TS; confers resistance to anti-cancer agent fluorodeoxyuridine);
human glutathione S-transferase alpha (GSTA1; conjugates
glutathione to the stem cell selective alkylator busulfan;
chemoprotective selectable marker in CD34+ cells); CD24 cell
surface antigen in hematopoietic stem cells; human CAD gene to
confer resistance to N-phosphonacetyl-L-aspartate (PALA); human
multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein
selectable by increased drug resistance or enriched by FACS); human
CD25 (IL-2.alpha.; detectable by Mab-FITC); Methylguanine-DNA
methyltransferase (MGMT; selectable by carmustine); rhamnose; and
Cytidine deaminase (CD; selectable by Ara-C). "Selective medium" as
used herein refers to cell growth medium to which has been added a
chemical compound or biological moiety that selects for or against
selectable markers.
[0056] The terms "target genomic DNA sequence", "target sequence",
or "genomic target locus" refer to any locus in vitro or in vivo,
or in a nucleic acid (e.g., genome or episome) of a cell or
population of cells, in which a change of at least one nucleotide
is desired using a nucleic acid-guided nuclease editing system. The
target sequence can be a genomic locus or extrachromosomal
locus.
[0057] The terms "transformation", "transfection" and
"transduction" are used interchangeably herein to refer to the
process of introducing exogenous DNA into cells.
[0058] A "vector" is any of a variety of nucleic acids that
comprise a desired sequence or sequences to be delivered to and/or
expressed in a cell. Vectors are typically composed of DNA,
although RNA vectors are also available. Vectors include, but are
not limited to, plasmids, fosmids, phagemids, virus genomes, BACs,
YACs, PACs, synthetic chromosomes, and the like. In some
embodiments, a coding sequence for a nucleic acid-guided nuclease
is provided in a vector, referred to as an "engine vector." In some
embodiments, the editing cassette may be provided in a vector,
referred to as an "editing vector." In some embodiments, the coding
sequence for the nucleic acid-guided nuclease and the editing
cassette are provided in the same vector.
Nuclease-Directed Genome Editing Generally
[0059] The compositions, methods, modules and automated instruments
described herein are employed to allow one to perform nucleic acid
nuclease-directed genome editing to introduce desired edits to a
population of live mammalian cells. The compositions and methods
entail creating reagent bundles (RBs) comprising many clonal (i.e.,
identical) copies of editing cassettes in a droplet formed in an
immiscible fluid carrier, followed by co-localizing the RBs with
live mammalian cells to effect editing of the genome of the
mammalian cells by the editing cassettes.
[0060] Generally, a nucleic acid-guided nuclease or nickase fusion
complexed with an appropriate synthetic guide nucleic acid in a
cell can cut the genome of the live cell at a desired location. The
guide nucleic acid helps the nucleic acid-guided nuclease or
nickase fusion recognize and cut the DNA at a specific target
sequence. By manipulating the nucleotide sequence of the guide
nucleic acid, the nucleic acid-guided nuclease or nickase fusion
may be programmed to target any DNA sequence for cleavage as long
as an appropriate protospacer adjacent motif (PAM) is nearby. In
certain aspects, the nucleic acid-guided nuclease or nickase fusion
editing system may use two separate guide nucleic acid molecules
that combine to function as a guide nucleic acid, e.g., a CRISPR
RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In other
aspects and preferably, the guide nucleic acid is a single guide
nucleic acid construct that includes both 1) a guide sequence
capable of hybridizing to a genomic target locus, and 2) a scaffold
sequence capable of interacting or complexing with a nucleic
acid-guided nuclease or nickase fusion.
[0061] In general, a guide nucleic acid (e.g., gRNA) complexes with
a compatible nucleic acid-guided nuclease or nickase fusion and can
then hybridize with a target sequence, thereby directing the
nuclease or nickase fusion to the target sequence. A guide nucleic
acid can be DNA or RNA; alternatively, a guide nucleic acid may
comprise both DNA and RNA. In some embodiments, a guide nucleic
acid may comprise modified or non-naturally occurring nucleotides.
Preferably and typically, the guide nucleic acid comprises RNA and
the gRNA is encoded by a DNA sequence on an editing cassette along
with the coding sequence for a donor DNA (e.g., whether a donor DNA
to recombine with the target or a donor DNA to be reverse
transcribed into the target region). Covalently linking the gRNA
and donor DNA allows one to scale up the number of edits that can
be made in a population of cells tremendously. Methods and
compositions for designing and synthesizing editing cassettes
(e.g., CREATE cassettes) are described in, e.g., U.S. Pat. Nos.
9,982,278; 10,266,849; 10,240,167; 10,351,877; 10,364,442;
10,435,715; 10,465,207; 10,669,559; 10,711,284; and 10,713,180 and
U.S. Ser. Nos. 16/550,092 and 16/938,739, all of which are
incorporated by reference herein. Alternatively, the nuclease or
nickase fusion enzyme and gRNAs may be introduced into the cells as
ribonuclease/protein complexes (RNPs), with the donor DNA
introduced as an oligonucleotide or sequence to be transcribed from
a vector. The donor DNA most often is introduced simultaneously
with the RNPs, but may also be introduced separately from the
RNPs.
[0062] A guide nucleic acid comprises a guide sequence, where the
guide sequence is a polynucleotide sequence having sufficient
complementarity with a target sequence to hybridize with the target
sequence and direct sequence-specific binding of a complexed
nucleic acid-guided nuclease or nickase fusion to the target
sequence. The degree of complementarity between a guide sequence
and the corresponding target sequence, when optimally aligned using
a suitable alignment algorithm, is about or more than about 50%,
60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal
alignment may be determined with the use of any suitable algorithm
for aligning sequences. In some embodiments, a guide sequence is
about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or
more nucleotides in length. In some embodiments, a guide sequence
is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in
length. Preferably the guide sequence is 10-30 or 15-20 nucleotides
long, or 15, 16, 17, 18, 19, or 20 nucleotides in length.
[0063] In general, to generate an edit in the target sequence, the
gRNA/nuclease or gRNA/nickase fusion complex binds to a target
sequence as determined by the guide RNA, and the nuclease or
nickase fusion recognizes a protospacer adjacent motif (PAM)
sequence adjacent to the target sequence. The target sequence can
be any polynucleotide endogenous or exogenous to the cell, or in
vitro. For example, in the case of mammalian cells the target
sequence is typically a polynucleotide residing in the nucleus of
the cell. A target sequence can be a sequence encoding a gene
product (e.g., a protein) or a non-coding sequence (e.g., a
regulatory polynucleotide, an intron, a PAM, a control sequence, or
"junk" DNA). The proto-spacer mutation (PAM) is a short nucleotide
sequence recognized by the gRNA/nuclease complex. The precise
preferred PAM sequence and length requirements for different
nucleic acid-guided nucleases or nickase fusions vary; however,
PAMs typically are 2-7 base-pair sequences adjacent or in proximity
to the target sequence and, depending on the nuclease or nickase,
can be 5' or 3' to the target sequence.
[0064] In most embodiments, genome editing of a cellular target
sequence both introduces a desired DNA change to a cellular target
sequence, e.g., the genomic DNA of a cell, and removes, mutates, or
renders inactive a proto-spacer mutation (PAM) region in the
cellular target sequence (e.g., thereby rendering the target site
immune to further nuclease binding). Rendering the PAM at the
cellular target sequence inactive precludes additional editing of
the cell genome at that cellular target sequence, e.g., upon
subsequent exposure to a nucleic acid-guided nuclease or nickase
fusion complexed with a synthetic guide nucleic acid in later
rounds of editing. Thus, cells having the desired cellular target
sequence edit and an altered PAM can be selected for by using a
nucleic acid-guided nuclease or nickase fusion complexed with a
synthetic guide nucleic acid complementary to the cellular target
sequence. Cells that did not undergo the first editing event will
be cut rendering a double-stranded DNA break, and thus will not
continue to be viable. The cells containing the desired cellular
target sequence edit and PAM alteration will not be cut, as these
edited cells no longer contain the necessary PAM site and will
continue to grow and propagate.
[0065] As for the nuclease or nickase fusion component of the
nucleic acid-guided nuclease editing system, a polynucleotide
sequence encoding the nucleic acid-guided nuclease or nickase
fusion can be codon optimized for expression in particular cell
types, such as bacterial, yeast, and, here, mammalian cells. The
choice of the nucleic acid-guided nuclease or nickase fusion to be
employed depends on many factors, such as what type of edit is to
be made in the target sequence and whether an appropriate PAM is
located close to the desired target sequence. Nucleases of use in
the methods described herein include but are not limited to Cas 9,
Cas 12/CpfI, MAD2, or MAD7, MAD 2007 or other MADzymes (see U.S.
Pat. Nos. 9,982,279; 10,337,028; 10,604,746; 10,665,114;
10,640,754, 10,876,102; 10,883,077; 10,704,033; 10,745,678;
10,724,021; 10,767,169; and 10,870,761 for sequences and other
details related to MADzymes). Nickase fusion enzymes typically
comprise a CRISPR nucleic acid-guided nuclease engineered to cut
one DNA strand in the target DNA rather than making a
double-stranded cut, and the nickase portion is fused to a reverse
transcriptase. For more information on nickases and nickase fusion
editing see U.S. Pat. No. 10,689,669 and U.S. Ser. Nos. 16/740,418;
16/740,420 and 16/740,421, both filed 11 Jan. 2020. Here, a coding
sequence for a desired nuclease or nickase fusion is typically on
an "engine vector" along with other desired sequences such as a
selective marker.
[0066] Another component of the nucleic acid-guided nuclease or
nickase fusion system is the donor nucleic acid comprising homology
to the cellular target sequence and an engineered change to the
cellular target sequence. For the present compositions, methods,
modules and instruments the donor nucleic acid typically is in the
same editing cassette as (e.g., is covalently-linked to) the guide
nucleic acid and is under the control of the same promoter as the
gRNA (that is, a single promoter driving the transcription of both
the editing gRNA and the donor nucleic acid). The donor nucleic
acid is designed to serve as a template for homologous
recombination with a cellular target sequence cleaved by the
nucleic acid-guided nuclease or the donor DNA serves as a template
to incorporate an edit into the target via reverse transcriptase
fused to a nickase as a part of the gRNA/nuclease complex. A donor
nucleic acid polynucleotide may be of any suitable length, such as
about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or
1000 nucleotides in length, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13 and up to 20 kb in length if combined with a dual gRNA
architecture as described in U.S. Pat. No. 10,711,284.
[0067] In certain preferred aspects, the donor nucleic acid can be
provided as an oligonucleotide of between 20-300 nucleotides, more
preferably between 50-250 nucleotides. The donor nucleic acid
comprises a region that is complementary to a portion of the
cellular target sequence (e.g., a homology arm(s)). When optimally
aligned, the donor nucleic acid overlaps with (is complementary to)
the cellular target sequence by, e.g., about as few as 4 (in the
case of nickase fusions) and as many as 20, 25, 30, 35, 40, 50, 60,
70, 80, 90 or more nucleotides (in the case of nucleases). The
donor nucleic acid typically comprises two homology arms (regions
complementary to the cellular target sequence) flanking the
mutation or difference between the donor nucleic acid and the
cellular target sequence, although in CREATE fusion embodiments,
the edit may be located at one end of a single homology arm rather
than positioned between two homology arms. The donor nucleic acid
comprises at least one mutation or alteration compared to the
cellular target sequence, such as an insertion, deletion,
modification, or any combination thereof compared to the cellular
target sequence.
[0068] As described in relation to the gRNA, the donor nucleic acid
is provided as part of a rationally-designed editing cassette along
with a promoter to drive transcription of both the gRNA and donor
DNA. As described below, the editing cassette may be provided as a
linear editing cassette, or the editing cassette may be inserted
into an editing vector. Moreover, there may be more than one, e.g.,
two, three, four, or more editing gRNA/donor nucleic acid pair
rationally-designed editing cassettes linked to one another in a
linear "compound cassette" or inserted into an editing vector;
alternatively, a single rationally-designed editing cassette may
comprise two to several editing gRNA/donor DNA pairs, where each
editing gRNA is under the control of separate different promoters,
separate promoters, or where all gRNAs/donor nucleic acid pairs are
under the control of a single promoter. In some embodiments the
promoter driving transcription of the editing gRNA and the donor
nucleic acid (or driving more than one editing gRNA/donor nucleic
acid pair) is an inducible promoter. In many if not most
embodiments of the compositions, methods, modules and instruments
described herein, the editing cassettes make up a collection or
library editing gRNAs and of donor nucleic acids representing,
e.g., gene-wide, pathway-wide or genome-wide libraries of editing
gRNAs and donor nucleic acids.
[0069] In addition to the donor nucleic acid, the editing cassettes
comprise one or more primer binding sites to allow for PCR
amplification of the editing cassettes. The primer binding sites
are used to amplify the editing cassette by using oligonucleotide
primers and may be biotinylated or otherwise labeled. In addition,
the editing cassette may comprise a barcode. A barcode is a unique
DNA sequence that corresponds to the donor DNA sequence such that
the barcode serves as a proxy to identify the edit made to the
corresponding cellular target sequence. The barcode typically
comprises four or more nucleotides. Also, in preferred embodiments,
an editing cassette or editing vector or engine vector further
comprises one or more nuclear localization sequences (NLSs), such
as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs.
Mammalian Cell Growth, Reagent Bundle Creation Transformation and
Editing
[0070] The present compositions and methods are drawn to nucleic
acid-guided nuclease editing of live mammalian cells. The methods
involve creating reagent bundle droplets comprising hundreds of
thousands to millions of clonal copies of an editing cassette in
each RB. A library of RB s comprises hundreds to thousands to tens
of thousands of different editing cassettes; thus, the methods are
scalable for delivering hundreds, thousands, tens of thousands or
more different editing cassettes to a population of live cells.
Once the library of RBs is created, the methods include delivering
or co-localizing the RBs with the live mammalian cells,
transfecting the cells and finally culturing the edited cells.
[0071] In the present methods, mammalian cells are often grown in
culture for several passages before entry into an automated
process. Cell culture is the process by which cells are grown under
controlled conditions, almost always outside the cell's natural
environment. For mammalian cells, culture conditions typically vary
somewhat for each cell type but generally include a medium and
additives that supply essential nutrients such as amino acids,
carbohydrates, vitamins, minerals, growth factors, hormones, and
gases such as, e.g., O.sub.2 and CO.sub.2. In addition to providing
nutrients, the medium typically regulates the physio-chemical
environment via a pH buffer, and most cells are grown at 37.degree.
C. Many mammalian cells require or prefer a surface or artificial
substrate on which to grow (e.g., adherent cells), whereas other
cells such as hematopoietic cells and some adherent cells can be
grown in or adapted to grow in suspension. Adherent cells often are
grown in 2D monolayer cultures in petri dishes or flasks, but some
adherent cells can grow in suspension cultures to higher density
than would be possible in 2D cultures. "Passages" generally refers
to transferring a small number of cells to a fresh substrate with
fresh medium, or, in the case of suspension cultures, transferring
a small volume of the culture to a larger volume of medium.
[0072] Mammalian cells include primary cells, which are cultured
directly from a tissue and typically have a limited lifespan in
culture; established or immortalized cell lines, which have
acquired the ability to proliferate indefinitely either through
random mutation or deliberate modification such as by expression of
the telomerase gene; and stem cells, of which there are
undifferentiated stem cells or partly-differentiated stem cells
that can both differentiate into various types of cells and divide
indefinitely to produce more of the same stem cells.
[0073] Primary cells can be isolated from virtually any tissue.
Immortalized cell lines can be created or may be well-known,
established cell lines such as human cell lines DU145 (derived from
prostate cancer cells); H295R (derived from adrenocortical cancer
cells); HeLa (derived from cervical cancer cells); KBM-7 (derived
from chronic myelogenous leukemia cells); LNCaP (derived from
prostate cancer cells); MCF-7 (derived from breast cancer cells);
MDA-MB-468 (derived from breast cancer cells); PC3 (derived from
prostate cancer cells); SaOS-2 (derived from bone cancer cells);
SH-SY5Y (derived from neuroblastoma cells); T-047D (derived from
breast cancer cells); TH-1 (derived from acute myeloid leukemia
cells); U87 (derived from glioblastoma cells); and the National
Cancer Institute's 60 cancer line panel NCI60; and other
immortalized mammalian cell lines such as Vero cells (derived from
African green monkey kidney epithelial cells); the mouse line
MC3T3; rat lines GH3 (derived from pituitary tumor cells) and PC12
(derived from pheochromocytoma cells); and canine MDCK cells
(derived from kidney epithelial cells).
[0074] Stem cells are of particular interest in the methods and
compositions described herein. Generally speaking, there are three
general types of mammalian stem cells: adult stem cells (ASCs),
which are undifferentiated cells found living within specific
differentiated tissues including hematopoietic, mesenchymal,
neural, and epithelial stem cells; embryonic stem cells (ESCs),
which in humans are isolated from a blastocyst typically 3-5 days
following fertilization and which are capable of generating all the
specialized tissues that make up the human body; and induced
pluripotent stem cells (iPSCs), which are adult stem cells that are
created using genetic reprogramming with, e.g., protein
transcription factors.
[0075] Once the cells of choice have been grown and passaged
several times--in some embodiments off-instrument--in a first step
the mammalian cells that are to be edited are transferred to an
automated instrument where the cells are grown in cell culture and
the growth of the cells is monitored. Monitoring is usually
performed by imaging the cells as described infra and/or by, e.g.,
measuring pH of the medium using a medium comprising a pH
indicator. As opposed to 2D culture of cells as described above,
the present methods envision culturing the cells in suspension.
Growing cells in suspension can be effected in various forms.
Adherent cells that typically are grown in 2D cultures when grown
in suspension often aggregate into "clumps." For example, some
iPSCs grow well as aggregates in suspension, and are most healthy
growing in aggregates of 50-300 microns in size, starting off as
smaller aggregates 30-50 microns in size. iPSCs are typically grown
in culture 3-5 days between passaging and the larger aggregates are
broken into smaller aggregates by filtering them, e.g., through a
cell strainer (e.g., a sieve) with a 37 micron filter. The iPSCs
can grow indefinitely in 3D aggregates as long as they are passaged
into smaller aggregates when the aggregates become approximately
300-400 microns in size.
[0076] An alternative to growing cells in 3D aggregates is growing
cells on microcarriers. Generally, microcarriers are nonporous
(comprised of pore sizes range from 0-20 nm), microporous
(comprised of pore sizes range from 20 nm-1 micron), and
macroporous (comprised of pore sizes range from 1-50 microns)
microcarriers comprising natural organic materials such as, e.g.,
gelatin, collagen, alginate, agarose, chitosan, and cellulose;
synthetic polymeric materials such as, e.g., polystyrene,
polyacrylates such as polyacrylamide, polyamidoamine (PAMAM),
polyethylene oxide (PEO/PEG), poly(N-isopropylacrylamide) (PNIPAM),
polycaprolactone (PCL), polylactic acid (PLA), and polyglycolic
acid (PGA); inorganic materials such as, e.g., silica, silicon,
mica, quartz and silicone; as well as mixtures of natural,
polymeric materials, cross-linked polymeric materials, and
inorganic materials etc. on which animal cells can grow.
Microcarriers useful in the methods herein typically range in size
from 30-1200 microns in diameter and more typically range in size
from 40-200 or from 50-150 microns in diameter.
[0077] Finally, another option for growing mammalian cells for
editing in the compositions, methods, modules and automated
instruments described herein is growing single cells in suspension
using a specialized medium such as that developed by ACCELLTA.TM.
(Haifa, Israel). Cells grown in this medium must be adapted to this
process over many cell passages; however, once adapted the cells
can be grown to a density of >40 million cells/ml and expanded
50-100.times. in approximately a week, depending on cell type.
[0078] There are three exemplary modules--as an alternative to
classic culture in flasks or plates--for growing and monitoring
cells off-instrument or in the automated instruments described
herein. One module is a rotating growth module, which is depicted
in FIGS. 3A-3E, another module is a tangential flow filtration
module, which is depicted in FIGS. 4A-4G and finally another module
is a bioreactor, features of which are depicted in FIGS. 5A-5I.
These modules can be adapted to dissociate cells (if required) as
well and are described in detail in relation to these figures. The
cells grown off-instrument or in a growth module of the automated
instrument as well as reagents needed for cell growth, nucleic acid
amplification, cell transfection or transduction, cell editing and
enrichment may be provided in a reagent cartridge. The cells and
reagents are moved from the reagent cartridge and between modules
by a robotic liquid handling system including the gantry. As an
example, the robotic liquid handling system may include an
automated liquid handling system such as those manufactured by
Tecan Group Ltd. of Mannedorf, Switzerland, Hamilton Company of
Reno, Nev. (see, e.g., WO2018015544A1 to Ott, entitled "Pipetting
device, fluid processing system and method for operating a fluid
processing system"), or Beckman Coulter, Inc. of Fort Collins,
Colo. (see, e.g., US20160018427A1 to Striebl et al., entitled
"Methods and systems for tube inspection and liquid level
detection"), and typically includes an air displacement
pipettor.
[0079] Reagent cartridges, such as those described in U.S. Pat. No.
10,376,889; 10,406,525; 10,478,222; 10,576,474; 10,639,637 and
10,738,271 allow for particularly easy integration with liquid
handling instrumentation. In some embodiments, only the air
displacement pipettor is moved by the gantry and the various
modules and reagent cartridge remain stationary. In alternative
embodiments, an automated mechanical motion system (actuator)
additionally supplies XY axis motion control or XYZ axis motion
control to one or more modules and/or cartridges of the automated
multi-module cell processing system. Used pipette tips, for
example, may be placed by the robotic handling system in a waste
repository. For example, an active module may be raised to come
into contact-accessible positioning with the robotic handling
system or, conversely, lowered after use to avoid impact with the
robotic handling system as the robotic handling system is moving
materials to other modules within the automated multi-module cell
processing instrument. Alternatively, the cells may be transferred
to the growth module by the user.
[0080] Alternatively, in some embodiments, a gantry and/or an air
displacement pump is not used; instead, in one embodiment reagents
are individually connected to the bioreactor, typically via tubing
or microfluidic circuits; in another embodiment, reagents may be
connected to a manifold that has a single connection to the
bioreactor. In some embodiments, the bioreactor is a completely
closed fluidic system; that is, e.g., no pipets piercing reagent
tubes and transferring liquid.
[0081] In a next step, the cells that have been grown in suspension
or on microcarriers are dissociated or, if grown on microcarriers,
may be dissociated from the microcarrier and/or transferred to
fresh microcarriers. Dissociation is required if the cells grown as
cell aggregates. In one embodiment, dissociation may be via
mechanical means such as agitation or by a filter, frit or sieve.
Such a filter, frit or sieve may be adapted to be part of the
rotating growth module, tangential flow filtration module, or
bioreactor module as described in relation to FIGS. 3A-3E, 4A-4E,
and 5A-5I or may be a separate "dissociation only module." As an
alternative, aggregates of cells may be dissociated by enzymes such
as hemagglutinin, collagenase, dispase and trypsin, which can be
added to the medium of the growing cells in the rotating growth
module, tangential flow filtration module or bioreactor. If the
cells are grown on microcarriers, the cells can be dissociated from
the microcarriers using enzymes that are typically used in cell
culture to dissociate cells in 2D culture, such as collagenase,
trypsin or pronase or by non-enzymatic methods including EDTA or
other chelating chemicals. In a bioreactor, dissociation can be
performed mechanically using, e.g., an impeller or enzymatically,
or cells grown in a bioreactor may be transferred to a dissociation
module.
[0082] In parallel with growing and preparing the cells for
transfection, reagent bundles (RBs) are created and once the cells
have been dissociated, the reagent bundles and cells are
co-localized. The RBs (droplets) comprise thousands to millions of
clonal copies of an editing cassette--either RNA or DNA editing
cassettes and in various configurations, e.g., dsDNA, ssDNA, ssRNA,
linear or circular templates.
[0083] Finally, in some methods and instruments, the population of
cells after editing are enriched for edited cells by, e.g.,
magnetic beads, antibiotic selection, co-edit selection, or FACS
sorting, all of which are described in detail infra.
Liquid Droplet Reagent Delivery for Editing Cells Grown in
Suspension
[0084] FIG. 1A depicts an exemplary workflow employing
liquid-partitioned (e.g., droplet) delivery for editing mammalian
cells grown in suspension. In this exemplary embodiment,
microfluidic droplet generation is used to clonally amplify editing
cassettes or editing vectors used to transform or transfect
mammalian cells. Microfluidic droplet generation using two
immiscible fluids (e.g., an aqueous solution and an oil) that meet
at intersecting microchannels with droplets being generated at a
junction between the two microfluidic channels has been known in
the art for several decades. The terms "droplet" and "emulsion" are
used interchangeably herein to refer to an aliquot of one fluid
(here, an aqueous solution) in an immiscible carrier fluid (e.g.,
an oil), with the carrier fluid substantially surrounding the
aqueous droplet. (See FIG. 1B.) For example, in 1984 Shaw Stewart
taught the use of a device to produce microfluidic emulsion
droplets from two immiscible fluids that meet at an intersecting
channel. (UK Patent Application No. 2097692 to Shaw Stewart.) Shaw
Stewart introduced the concept of a microfluidic "T-junction" at
which droplets of an aqueous solution can be formed using a
continuously flowing immiscible "carrier phase." By the 1990s,
various groups were looking to both miniaturize and automate
biological and chemical reactions and by the early 2000s research
groups had shown proof of principle for the use of aqueous droplets
in an oil carrier for carrying out various analyses, cell sorting
operations and biochemical reactions including PCR. (See, e.g., US
Pub. No. 2002/0058332 to Quake, et al.)
[0085] The immiscible fluids used in the microchannels may be
provided by on-chip wells or off-chip reservoirs and the channels
intersect as T-junctions to form the droplets. A pressure
differential is used to control the flow of the fluids at the
T-junction, shearing-off of the aqueous fluid into the immiscible
oil flow to create droplets. By adjusting the pressure of the
flowing fluids, a pressure difference can be established to shear
off droplets of the aqueous solution at a regular frequency as the
aqueous solution enters the oil stream, thereby forming droplets in
the oil stream. The droplets optionally may be combined with other
droplets to effect a reaction between the reactants when the
droplets combine.
[0086] In the 2000s a "cross junction" strategy was introduced. In
the cross-junction approach, aqueous droplets are formed at
converging flows of the immiscible fluids where a continuous phase
(here, the oil phase) where the converging flows "pinch" off the
droplets of the aqueous phase at a droplet-forming junction. This
"flow focusing" approach, in which the aqueous droplet is focused
by the oil phase at the cross junction, is described, for example,
by Higuchi, et al. (US Pat. Pub. 2004/0068019) (See, e.g., FIG. 1B
infra.) Microfluidic devices or "chips" are available commercially
through, e.g., microfluidic ChipShop.TM. GmbH (Jena, Germany);
uFluidix.TM. (Toronto, Canada); Microflexis.TM. (Hamburg, Germany);
and microLIQUID.TM. (Gipuzkoa, Spain).
[0087] The present embodiment using droplets is particularly useful
in providing discretely-apportioned "mini reactors" and for
co-partitioning reagents with cells for further reaction and
analysis. Co-partitioning of reagents and sample components can be
used, for example, for reducing the complexity of sample material
by segregating portions of the sample into different partitions. As
with the gel beads described infra, by segregating reagents each
sample portion (e.g., cell or group of cells) can be subjected into
a different reaction with thousands, tens of thousands, hundreds of
thousands or more reactions taking place in parallel.
[0088] Further in the case of droplets in an emulsion, allocating
individual molecules (e.g., Poisson distribution of molecules in
droplets) is accomplished by introducing an aqueous stream of fluid
comprising the molecules into a flowing stream of a carrier fluid
such that droplets are generated at the junction of the two
streams. By providing the aqueous molecule-containing (or
cell-containing) stream at a certain concentration level, the
number of droplets containing molecules (or cells) can be
controlled. In the present methods, it is desirable to control the
relative flow rates of the aqueous and non-aqueous fluid such that,
on average, the droplets contain less than one molecule per droplet
to ensure that any one droplet will comprise either one molecule or
no molecule. Thus, the droplet method (or gel bead method) allows
one to partition a single editing cassette construct, amplify the
single copy to make thousands, tens of thousands, hundreds of
thousands or more copies for delivery as opposed to the other
method of pooling editing cassettes, amplifying the pooled editing
cassettes and then de-multiplexing the pool of amplified editing
cassettes. Both methods, however, result in RBs with hundreds of
thousands to millions of clonal copies of editing cassettes in or
on a partition (e.g., droplet, gel bead or microcarrier).
[0089] In addition, cell sorting may be performed on a microfluidic
device. While conventional methods can provide high efficiency
sorting in short timescales, advances in microfluidics have enabled
the realization of miniaturized devices offering similar
capabilities that exploit a variety of physical principles. These
technologies may be classified as either active or passive. Active
systems generally use external fields (e.g., acoustic, electric,
magnetic, and optical) to impose forces to displace cells for
sorting; whereas passive systems use inertial forces, filters, and
adhesion mechanisms to purify cell populations. Cell sorting on
microchips provides numerous advantages over conventional methods
by reducing the size of necessary equipment, eliminating
potentially biohazardous aerosols, and simplifying the complex
protocols commonly associated with cell sorting. Further, microchip
devices are well suited for parallelization, enabling complete
lab-on-a-chip devices for cellular isolation, analysis, and
experimental processing. Cell sorting on microchips includes
label-based sorting, detectable by electrokinetic mechanisms such
as, e.g., electrophoresis and dielectric phoresis;
acoutstrophoresis; optical sorting; mechanical systems; bead-based
sorting via, e.g., magnetophoresis and electrokinetic mechanisms;
and label-free cell sorting via, e.g., acoustophoresis and optical
switching mechanisms. Cell sorting in microfluidics devices is
discussed in, e.g., Shields, et al., Microfluidic Cell Sorting: A
Review of the Advances in the Separation of Cells from Debulking to
Rare Cell Isolation, Lab Chip, 15(5):1230-49 (2015); and U.S. Pub.
Nos. 2015/0268244; 2008/0213821; 2020/0190488; and
2005/0164158.
[0090] FIG. 1A shows that the liquid droplet workflow begins with
cell culture, typically off-instrument. Again, the cells may be
grown off-instrument in 2D culture in plates or tissue culture
flasks, or in 3D culture (if the cells are viable when grown in or
adapted to 3D culture) on microcarriers in spinner flasks or in a
bioreactor as described herein infra. As described previously, if
necessary, the cells are dissociated and added to medium in an
on-instrument growth module such as, e.g., a rotating growth vial,
TFF reservoir or bioreactor comprising cell growth medium such as
DMEM. If the cells are grown initially on cell growth
microcarriers, the microcarriers are transferred to a rotating
growth vial or TFF reservoir or bioreactor comprising cell growth
medium such as MEM, DMEM or the like and additional microcarriers.
Alternatively, single cells may be grown in suspension using the
specialized medium developed by ACCELLTA.TM. (Haifa, Israel);
however, cells grown in this medium must be adapted to this
process. Once grown, approximately 1e7 cells are transferred to the
automated instrument for growth. The cells are grown in 3D culture
in aggregates or on microcarriers in the RGV, TFF or bioreactor
for, e.g., three days or until a desired number of cells, e.g., 1e8
cells are present. During this growth cycle, the cells are
monitored for cell number, pH, and optionally other parameters.
[0091] Cell dissociation, if required, is performed by removing
cells from cell growth microcarriers using enzymes such as
collagenase, trypsin or pronase, or by non-enzymatic methods
including EDTA or other chelating chemicals, and then passing cell
aggregates through a filter, frit or sieve by agitating the cells
or if grown in suspension as aggregates simply by passing the
aggregates through a filter or sieve, or by treatment with, e.g.,
hemagluttinin. Alternatively, and as described in relation to FIG.
5D and Examples VIII and IX, cells grown on microcarriers in a
bioreactor can be detached and dissociated using an impeller, which
creates turbulence in the medium in which the cells are grown. In
addition, combination of these techniques may be used.
[0092] In the embodiments described herein, reagents are "packaged"
and delivered to the cells via a microfluidic device, described in
detail infra with reference to FIGS. 1B and 1C, and editing takes
place in droplets on the microfluidic device as well. FIG. 1B
depicts a microfluidic channel structure 100 for the flow focusing
or partitioning of individual molecules in droplets. The channel
structure 100 includes channel sections 102, 104, 106 and 108 all
joined at channel junction 110. In operation, an aqueous fluid
112--here, e.g., a buffer comprising PCR reagents (e.g., primers,
enzyme, dNTPs) that includes CREATE editing cassettes 114 at a
concentration determined to allow formation of droplets comprising
no editing cassette or one editing cassette--is transported along
channel section 102 into channel junction 110, while a second fluid
116 that is immiscible with aqueous fluid 112 is delivered to
channel junction 110 from channel sections 104 and 106 to create
droplets 118 and 120. In the present example, the concentration of
editing cassettes is such that at droplet formation, some droplets
contain an editing cassette while some droplets do not. Such a
Poisson distribution of editing cassettes in droplets allows for
clonal amplification of editing cassettes in the droplets where
each droplet then comprises many copies of a single editing
cassette or no editing cassettes. Channel section 108 may be
fluidically coupled to an outlet reservoir into which the droplets
may be stored.
[0093] The droplets comprising the editing cassettes and PCR
reagents described herein are characterized by having extremely
small volumes, e.g., approximately equal to or less than 10 nL (and
as low as 0.1 nL), approximately equal to or less than 5 nL,
approximately equal to or less than 2 nL, approximately equal to or
less than 1 nL, approximately equal to or less than 900 picoliters
(pL), approximately equal to or less than 800 pL, approximately
equal to or less than 700 pL, approximately equal to or less than
600 pL, approximately equal to or less than 500 pL, approximately
equal to or less than 400 pL, approximately equal to or less than
300 pL, approximately equal to or less than 200 pL, approximately
equal to or less than 100 pL, approximately equal to or less than
50 pL, approximately equal to or less than 25 pL, approximately
equal to or less than 10 pL, approximately equal to or less than 5
pL, approximately equal to or less than 1 pL, approximately equal
to or less than 500 nanoliters (fL), approximately equal to or less
than 100 fL, approximately equal to or less than 50 fL, or even
less.
[0094] The fluid that is immiscible with the aqueous fluid is
typically a non-polar hydrophobic fluid such as an oil, e.g.,
mineral oil, or an organic liquid such as hexadecane. Fluorinated
oils are commonly used, with a fluorosurfactant added to stabilize
the droplets that are formed. Non-limiting examples of fluorophilic
components that can be used in either a surfactant and/or a
continuous phase include: perfluorodecalin, perfluoromethyldecalin,
perfluoroindane, perfluorotrimethyl bicyclo[3.3.1]nonane,
perfluoromethyl adamantine, perfluoro-2,2,4,4-tetra-methylpentane;
9-12C perfluoro amines, e.g., perfluorotripropyl amine,
perfluorotributyl amine, perfluoro-1-azatricyclic amines;
bromofluorocarbon compounds, e.g., perfluorooctyl bromide and
perfluorooctyl dibromide; F-4-methyl octahydroquinolidizine and
perfluoro ethers, including chlorinated polyfluorocyclic ethers,
perfluoro-4-methylmorpholine, perfluorotriethylamine,
perfluoro-2-ethyltetrahydrofuran, perfluoro-2-butyltetrahydrofuran,
perfluoropentane, perfluoro(2-methylpentane), perfluorohexane,
perfluoro-4-isopropylmorpholine, perfluorodibutyl ether,
perfluoroheptane, perfluorooctane, perfluorotripropylamine,
perfluorononane, perfluorotributylamine, perfluorodihexyl ether,
perfluoro [2-(diethylamino)ethyl-2-(N-morpholino)ethyl]ether,
n-perfluorotetradecahydrophenanthrene, and mixtures thereof.
[0095] Generally, the channel sections are coupled to different
fluid sources, receiving and/or output reservoirs, tubing,
manifolds, compressors, pumps, vacuums, actuators or other flow
controls. For exemplary microfluidic droplet generators and
methods, see, e.g., U.S. Pat. Nos. 8,822,148; 8,304,193; 8,889,083;
9,216,392; 9,347,059; 9,089,844; 9,126,160; 9,500,664; 9,636,682;
and 9,649,635; and US Pub. Nos. 2010/0105122; 2014/0155295;
2018/0312873; 2019/0176152; and 2019/0233878.
[0096] In the present methods, the "payload" for the droplets is an
editing cassette construct, again where the vast majority of beads
comprise one or no editing cassette construct. As described above,
an editing cassette (e.g., CREATE cassette or CREATE fusion
cassette) comprises a coding sequence for transcription of a guide
nucleic acid or gRNA covalently linked to a coding sequence for
transcription of a donor DNA (whether configured for nuclease or
nickase fusion (e.g., CREATE fusion) editing) and an editing
cassette construct further comprises the editing cassette linked to
a promoter to drive transcription of the gRNA and donor DNA.
Covalently linking the gRNA and donor DNA allows one to scale up
the number of edits that can be made simultaneously in a population
of cells tremendously. In addition to the promoter, coding sequence
for transcription of the gRNA and coding sequence for transcription
of the donor DNA, the editing cassette construct may comprise other
functional elements that are used in processing (e.g.,
amplification) of the editing cassette sequences, including, e.g.,
primer sequences (targeted or universal, preferably in most
embodiments herein, universal), labels, enrichment sequences,
immobilization sequences, barcodes, and NLSs. (See, e.g., FIG. 11.)
The editing cassette construct can be, depending on the embodiment,
a linear construct, a circular construct, a double-stranded
construct or single-stranded construct and comprise DNA, RNA,
nucleic acid mimetics of a combination thereof.
[0097] FIG. 1C depicts an exemplary method for transforming and
editing live mammalian cells via nucleic acid-guided nuclease or
nickase fusion editing using the concept depicted in FIG. 1B. The
method depicted in FIG. 1C begins, at top left, with a pool of
CREATE editing cassette constructs. The pool of editing cassettes
is diluted in an aqueous solution comprising amplification reagents
(e.g., dNTPs, primers and polymerase enzyme) and introduced into a
microfluidic droplet generator device where aqueous droplets
comprising a Poisson distribution of editing cassette constructs
are generated, such as seen in FIG. 1B. In the presence of the
amplification reagents, the editing cassette constructs in the
droplets comprising editing cassette constructs are amplified,
creating clonal copies of the editing cassette constructs confined
to the droplets. In the droplets that did not receive an editing
cassette, there should be no amplification. Thermocycling is
provided if PCR is performed as opposed to isothermal
amplification.
[0098] The editing cassette constructs, the primers or dNTPs used
to amplify the editing cassette constructs may comprise a label,
such as a fluorescent or chemiluminescent label, which labels the
amplicons as they are produced. In certain cases, the detectable
label is a fluorophore, which, after absorption of energy, emits
radiation at a defined wavelength. Fluorescent detectable labels
include, for example, dansyl-functionalized fluorescent moieties
(see, e.g., Welch et al., Chem. Eur. J. 5(3):951-960 (1999)); and
fluorescent labels Cy3 and Cy5 (see, e.g., Zhu et al., Cytometry
28:206-211 (1997)). Detectable labels are also disclosed in Prober
et al., Science 238:336-341 (1987); Connell et al., BioTechniques
5(4):342-384 (1987); Ansorge et al., Nucl. Acids Res.
15(11):4593-4602 (1987) and Smith et al., Nature 321:674 (1986).
Other commercially available fluorescent labels include, but are
not limited to, fluorescein, rhodamine (including TMR, Texas red
and Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene
and the cyanins.
[0099] Following PCR, the droplets are, e.g., FACS sorted where
droplets comprising amplified editing cassette constructs are
segregated from droplets that do not contain amplified editing
cassette constructs. This process is shown in FIG. 1C at top right.
For sorting, the droplets may be sorted suspended in the oil
carrier or the droplets in the oil carrier may be further dispersed
in an aqueous phase--creating a double emulsion--for sorting.
[0100] Once sorted, the segregated droplets comprising the
amplified editing cassette constructs can then be introduced into a
stream of carrier fluid comprising aqueous droplets containing,
e.g., transformation, transfection or transduction reagents such
as, for example, lipofection, nucleofection, biolistic, virosome,
liposome, immunoliposome, polycation or lipid:nucleic acid
conjugate reagents. The step of combining or coalescing droplets
comprising amplified editing cassettes and droplets comprising
lipofection reagents is depicted in FIG. 1C at bottom left. The
droplets comprising the amplified editing cassettes and
transformation or transfection reagents are coalesced in the
microfluidic device using one of several methods that may be used
to cause coalescence of droplets. Such methods include providing a
localized electric field to coalesce the droplet, providing locally
a chemical that disrupts or destabilizes the surfactant in the
continuous phase, such as, e.g., perfluoro-octanol, or use of a
textured surface in the flow path of the microfluidic channel as
described in US Pub. No. 2019/0176152.
[0101] Following coalescence of the amplified editing cassette
constructs and transformation or transfection reagents into single
partitions, droplets or "reagent bundles", the reagent bundles may
then be introduced into a stream of aqueous droplets comprising one
or more cells in each droplet. This process is depicted in FIG. 1C
at bottom right. Again, the cells are grown on-instrument in one of
the three growth modules described infra. In this step, the reagent
bundles are coalesced with the droplets comprising cells to effect
transfection of the editing cassette constructs into cells. Once
sufficient time for transfection has occurred, for example, from 1
minute to 24 hours, or from 2 minutes to 12 hours, or from 5
minutes to 8 hours, or from 7 minutes to 4 hours, or from 10
minutes to 2 hours, the droplets containing the cells and reagent
bundles can be demulsified, and the cells are allowed to continue
to grow in an appropriate medium or the cells optionally are washed
and plated or washed and cultured in 3D culture in an appropriate
medium (e.g., on microcarriers in the RGV, TFF or bioreactor) which
allows the cells to recover and grow.
[0102] In the embodiment shown in FIG. 1C (and in FIG. 1D), a
vector comprising the coding sequence for the nuclease has already
been transformed or transfected into the cells prior to dispersing
the cells in droplets in an oil carrier. However, in alternative
embodiments, the nuclease may be integrated into the genome of the
cells to be edited or a coding sequence for the nuclease or
nuclease fusion (either as a linear construct or as part of an
engine vector) or the nuclease or nuclease fusion protein itself
may be provided in droplets with the transformation or transfection
reagents and transfected into the cells concurrently with the
amplified editing cassettes.
Solid Wall Partitioned Delivery for Editing Cells Grown in
Suspension
[0103] FIG. 2A depicts an exemplary workflow employing solid wall
partitioning and editing of mammalian cells grown in suspension.
Again, the cells may be grown off-instrument in 2D culture in
plates or tissue culture flasks, or in 3D culture (if the cells are
viable when grown in or adapted to 3D culture) on microcarriers in
spinner flasks or in a bioreactor as described herein infra. As
described previously, if the cells are initially grown in 2D or 3D
culture, the cells are dissociated and added to medium in, e.g., a
rotating growth vial, TFF reservoir or bioreactor comprising cell
growth medium or SWIIN reservoir 652 in FIG. 6A. The overall work
flow for cell growth in SWIIN comprises loading a cell culture to
be grown into a reservoir 652, preferably bubbling air or an
appropriate gas through the cell culture, passing or flowing the
cell culture to reservoir 654 by modifying reservoir seal 662 into
a connection channel, optionally adding additional fresh or
different medium to the cell culture and optionally bubbling air or
gas through the cell culture, then repeating the process, all while
measuring, e.g., the optical density of the cell culture in the
reservoirs continuously or at desired intervals. Again, cell growth
monitoring can be performed by imaging, for example, by allowing
the microcarriers to settle and imaging the bottom of the SWIIN
reservoir. Alternatively, an aliquot of the culture is removed and
run through a flow cell for imaging.
[0104] In yet another alternative, the cells may express a
fluorescent protein and fluorescence is measured. In an alternative
approach, reservoir seal 662 is modified into a connection channel
containing a filter, frit or sieve for passaging large cell
aggregates into smaller aggregates or clumps.
[0105] If the cells are grown initially on microcarriers, the
microcarriers are transferred to a rotating growth vial, TFF
reservoir, or bioreactor comprising cell growth medium and,
optionally, additional microcarriers comprising cell growth medium
and additional microcarriers. Alternatively, single cells may be
grown in suspension using the specialized medium developed by
ACCELLTA.TM. (Haifa, Israel). Once grown, approximately 1e7 cells
are transferred to the automated instrument for growth. During this
growth cycle, the cells are monitored for cell number, pH, and
optionally other parameters. Cell dissociation, if required, is
performed by removing cells from microcarriers using enzymes such
as collagenase, trypsin or pronase, or by non-enzymatic methods
including EDTA or other chelating chemicals, and then passing cell
aggregates through a filter, frit or sieve or by using another
mechanical means such as an impeller or other agitation means.
[0106] In this embodiment, like the liquid droplet embodiment,
reagents are "packaged" in a microfluidic device; however, unlike
the liquid droplet embodiment, reagents are delivered to the cells
in a solid wall device. Thus, the cells, once grown on-instrument
and dissociated, are dispensed into microwells, such as the solid
wall isolation, incubation, and normalization (SWIIN) device
depicted in FIGS. 6A-6D and described in detail below, such that
each well or partition (in some embodiments, at least 100,000
partitions) comprises approximately 250 to 1500 cells, or from 500
to 1000 cells. Exemplary processes for forming gel beads are
described in relation to FIG. 2B-2D infra.
[0107] FIG. 2B depicts an exemplary method for encapsulating
reagent bundles comprising amplified editing cassette constructs in
gel beads. At top of FIG. 2B, an aqueous solution comprising PCR
reagents is flowed through a channel 202, and an aqueous solution
comprising editing cassette constructs 212 at a dilution
pre-determined to provide droplets with one or no editing cassette
is flowed through a channel 204 that intersects the channel with
the PCR reagents (e.g., enzyme, primers and dNTPs) 202. The channel
with the combined editing cassette constructs and PCR reagents is
then flow-focused by two converging streams (from channels 206 and
208) of an oil carrier, thereby forming droplets of PCR reagents
and editing cassette constructs 218 or droplets containing PCR
reagents only 220. At bottom of FIG. 2B shows a droplet 218
comprising PCR reagents and an editing cassette construct 212. In a
next step, the droplet is polymerized to form a gel bead or gel
matrix 222. In some embodiments, a gel bead can create a system
comprising a large molecule such as an editing cassette construct
within the boundary of the gel bead, while allowing small molecules
such as enzymes dNTPs, primers and, e.g., lipoplex, to permeate the
matrix. Further, the gel bead may comprise a labile bond such that
after the gel bead is degraded, the large molecule is released from
the gel matrix.
[0108] In some of the embodiments herein--such as the method
depicted in this FIG. 2B--the editing cassette constructs, nuclease
or nickase fusion coding sequence, engine vector, PCR reagents,
transformation or transfection reagents, etc. are encapsulated in
gel beads during gel bead generation (e.g., during polymerization
of precursors). In other embodiments, small molecules such as
lipofect or PCR reagents (e.g., primers, polymerases, dNTPs,
co-factors) and buffers may be added to the gel beads after
formation. Alternatively, the polymer network may be chemically
modified to conjugate specifically with a target molecule for
retention. Encapsulated reagents and molecules may be released from
a gel bead upon degradation of the gel bead.
[0109] Looking again at FIG. 2B, following polymerization (e.g.,
formation) of the gel bead, amplification of editing cassette
constructs 212 takes place inside the gel bead 224. Alternatively,
however, amplification of the editing cassette constructs 212 may
take place before gel bead polymerization; that is, the
amplification take place in the droplet before polymerization into
a gel bead.
[0110] FIG. 2C is similar to FIG. 1C at top; however, in FIG. 2C
the editing cassettes, PCR reagents and transfection reagents are
combined into a gel bead rather than retained within a droplet. The
method depicted in FIG. 2C begins--like the method depicted in FIG.
1B--with a pool of editing cassette constructs in an aqueous
solution comprising PCR reagents and, here, transfection reagents.
The pool of editing cassettes and reagents is introduced into a
microfluidic droplet generator device such as those described supra
where aqueous droplets comprising a Poisson distribution of editing
cassette constructs are generated. Here, however, the generated
aqueous droplets are subsequently polymerized into gel beads. Once
the gel beads are formed, the editing cassette constructs in the
gel beads are amplified thereby creating clonal copies of the
editing cassette constructs in the gel beads. Again, in the
droplets that did not receive an editing cassette construct, there
should be no amplification. As with the method depicted in FIG. 1C,
the editing cassette constructs, primers used to amplify the
editing cassette constructs or dNTPs may comprise a label, such as
a fluorescent or chemiluminescent label, which labels the
amplicons. Once amplification has taken place the gel beads are
demulsified (e.g., removed from the carrier fluid by washing or
methods such as chemical destabilization, mechanical rupture, or
electrocoalescence) and are sorted to separate the editing cassette
construct-containing gel beads from the gel beads that did not
receive an editing cassette during droplet formation.
[0111] Again, cell sorting on microfluidic devices has been
accomplished by several different technologies, generally
classified as either active or passive. Active systems generally
use external fields (e.g., acoustic, electric, magnetic, and
optical) to impose forces to displace cells for sorting; whereas
passive systems use inertial forces, filters, and adhesion
mechanisms to purify cell populations. Cell sorting on microchips
provides numerous advantages over conventional methods by reducing
the size of necessary equipment, eliminating potentially
biohazardous aerosols, and simplifying the complex protocols
commonly associated with cell sorting. Further, microchip devices
are well suited for parallelization, enabling complete
lab-on-a-chip devices for cellular isolation, analysis, and
experimental processing. Cell sorting on microchips includes
label-based sorting, detectable by electrokinetic mechanisms such
as, e.g., electrophoresis and dielectric phoresis;
acoutstrophoresis; optical sorting; mechanical systems; bead-based
sorting via, e.g., magnetophoresis and electrokinetic mechanisms;
and label-free cell sorting via, e.g., acoustophoresis and optical
switching mechanisms. Cell sorting in microfluidics devices are
discussed in, e.g., Shields, et al., Microfluidic Cell Sorting: A
Review of the Advances in the Separation of Cells from Debulking to
Rare Cell Isolation, Lab Chip, 15(5):1230-49 (2015); and U.S. Pub.
Nos. 2015/0268244; 2008/0213821; 2020/0190488; and
2005/0164158.
[0112] FIG. 2D depicts a method for delivering the gel beads to
cells for transfection or transformation. First, cells are provided
on a substrate, partitioned into wells or other partitions. Next,
gel beads are distributed into the wells or partitions such that
there is one gel bead per partition, which can be controlled by the
size of the partition and the gel bead. The gel bead is then
dissolved or disrupted thereby delivering the contents or "payload"
of the gel bead (e.g., the amplified editing cassette constructs
and transformation or transfection reagents) to the cells.
[0113] The gel beads used in the method depicted in FIG. 2D are
dissolvable or degradable upon exposure to one or more stimuli; for
example, pH changes, a change in temperature, exposure to light, or
exposure to a certain chemical species such as a reducing agent.
For example, a degradable gel bead may comprise one or more species
with a labile bond such that, when the gel bead is exposed to the
appropriate stimuli, the labile bond is broken, and the bead
degrades. The labile bond may be a chemical bond or another type of
physical interaction such as, e.g., van der Waals interaction,
dipole-dipole interaction, or the like. In some cases, the
crosslinker used to generate a bead may comprise a labile bond,
where, upon exposure to the appropriate conditions, the labile bond
can be broken and the gel bead degraded. For example, upon exposure
of a polyacrylamide gel bead comprising disulfide crosslinkers to a
reducing agent, the disulfide bonds can be broken and the bead
degraded. Examples of reducing agents include
.beta.-mercaptoethanol, dithiothreitol (DTT),
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),
tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
Thus, in the method depicted in FIG. 6D, a reducing agent may be
delivered to the substrate on which the cells and gel beads have
been distributed to dissolve the gel bead.
Cell Growth Modules
Rotating Growth Module
[0114] As described above, the mammalian cells of choice are
initially cultured off-instrument to obtain a suitable number of
cells for growth and transfection. The cells are then transferred
on instrument to a growth module, three of which are described
herein. One growth module is a rotating growth module depicted and
described in relation to FIGS. 3A-3E. FIG. 3A shows one embodiment
of a rotating growth vial 300 for use with a cell growth module and
in the automated multi-module cell processing instruments described
herein. The rotating growth vial 300 is an optically-transparent
container having an open end 304 for receiving liquid media and
cells, a central vial region 306 that defines the primary container
for growing cells, a tapered-to-constricted region 318 defining at
least one light path 310, a closed end 316, and a drive engagement
mechanism 312. The rotating growth vial 300 has a central
longitudinal axis 320 around which the vial rotates, and the light
path 310 is generally perpendicular to the longitudinal axis of the
vial. The first light path 310 is positioned in the lower
constricted portion of the tapered-to-constricted region 318.
Optionally, some embodiments of the rotating growth vial 300 have a
second light path 308 in the tapered region of the
tapered-to-constricted region 318. Both light paths in this
embodiment are positioned in a region of the rotating growth vial
that is constantly filled with the cell culture (cells+growth
media) and are not affected by the rotational speed of the growth
vial. The first light path 310 is shorter than the second light
path 308 allowing for, e.g., sensitive measurement of OD values
when the OD values of the cell culture in the vial are at a high
level (e.g., later in the cell growth process), whereas the second
light path 308 allows for, e.g., sensitive measurement of OD values
when the OD values of the cell culture in the vial are at a lower
level (e.g., earlier in the cell growth process).
[0115] The drive engagement mechanism 312 engages with a motor (not
shown) to rotate the vial. In some embodiments, the motor drives
the drive engagement mechanism 312 such that the rotating growth
vial 300 is rotated in one direction only, and in other
embodiments, the rotating growth vial 300 is rotated in a first
direction for a first amount of time or periodicity, rotated in a
second direction (i.e., the opposite direction) for a second amount
of time or periodicity, and this process may be repeated so that
the rotating growth vial 300 (and the cell culture contents) are
subjected to an oscillating motion. Further, the choice of whether
the culture is subjected to oscillation and the periodicity
therefor may be selected by the user. The first amount of time and
the second amount of time may be the same or may be different. The
amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,
2, 3, 4 or more minutes. In another embodiment, in an early stage
of cell growth the rotating growth vial 300 may be oscillated at a
first periodicity (e.g., every 60 seconds), and then a later stage
of cell growth the rotating growth vial 300 may be oscillated at a
second periodicity (e.g., every one second) different from the
first periodicity.
[0116] The rotating growth vial 300 may be reusable or, preferably,
the rotating growth vial is consumable. In some embodiments, the
rotating growth vial is consumable and is presented to the user
pre-filled with growth medium, where the vial is hermetically
sealed at the open end 304 with a foil seal. A medium-filled
rotating growth vial packaged in such a manner may be part of a kit
for use with a stand-alone cell growth device or with a cell growth
module that is part of an automated multi-module cell processing
system. To introduce cells into the vial, a user need only pipette
up a desired volume of cells and use the pipette tip to punch
through the foil seal of the vial. Open end 304 may optionally
include an extended lip 302 to overlap and engage with the cell
growth device. In automated systems, the rotating growth vial 300
may be tagged with a barcode or other identifying means that can be
read by a scanner or camera (not shown) that is part of the
automated system.
[0117] The volume of the rotating growth vial 300 and the volume of
the cell culture (including growth medium) may vary, but the volume
of the rotating growth vial 300 must be large enough to generate a
specified total number of cells. In practice, the volume of the
rotating growth vial 300 may range from 5-1000 mL, 10-500 mL, or
from 20-250 mL. Likewise, the volume of the cell culture
(cells+growth media) should be appropriate to allow proper aeration
and mixing in the rotating growth vial 300. Proper aeration
promotes uniform cellular respiration within the growth medium.
Thus, the volume of the cell culture should be approximately 5-85%
of the volume of the growth vial or from 20-60% of the volume of
the growth vial. For example, for a 300 mL growth vial, the volume
of the cell culture would be from about 15 mL to about 260 mL, or
from 6 mL to about 180 mL.
[0118] The rotating growth vial 300 preferably is fabricated from a
bio-compatible optically transparent material--or at least the
portion of the vial comprising a light path for imaging is
transparent. Additionally, material from which the rotating growth
vial is fabricated should be able to be cooled to about 4.degree.
C. or lower and heated to about 55.degree. C. or higher to
accommodate both temperature-based cell assays and long-term
storage at low temperatures. Further, the material that is used to
fabricate the vial must be able to withstand temperatures up to
55.degree. C. without deformation while spinning. Suitable
materials include cyclic olefin copolymer (COC), glass, polyvinyl
chloride, polyethylene, polyetheretherketone (PEEK), polypropylene,
polycarbonate, poly(methyl methacrylate) (PMMA), polysulfone,
poly(dimethylsiloxane), and co-polymers of these and other
polymers. Preferred materials include polypropylene, polycarbonate,
or polystyrene. In some embodiments, the rotating growth vial is
inexpensively fabricated by, e.g., injection molding or
extrusion.
[0119] FIG. 3B illustrates a top view of a rotating growth vial
300. In some examples, the vial 300 may include one or more fins or
paddles 322 affixed to an inner surface of the vial wall, where the
paddles protrude toward the center of the vial 300. The vial 300
shown in FIG. 3B includes three paddles 322 that are substantially
equally spaced around the periphery of the vial 300, but in other
examples vial 300 may include two, four, or more paddles 322. The
paddles, in some implementations, provide increased mixing and
aeration within the vial 300 rotating within a cell growth device,
which facilitates cell growth. In other configurations, there may
be concentric rows of raised features disposed on the inner surface
of the rotating growth vial and the features may be arranged
horizontally or vertically; and in other aspects, there may be a
spiral configuration of raised features disposed on the inner
surface of the rotating growth vial. In alternative aspects, the
fins or paddles or concentric rows of raised features may be
disposed upon a post or center structure of a rotating growth vial,
where the paddles or features radiate out from the center of the
vial toward the inner walls of the vial. In some aspects, the width
of the paddles or interior features varies with the size or volume
of the rotating growth vial, and may range from 1/8 to just under
1/2 the radius of the rotating growth vial, or from 1/4 to 1/3 the
radius of the rotating growth vial. The length of the paddles
varies with the size or volume of the rotating growth vial and may
range from 1/4 to 4/5 the length of the rotating growth vial, or
from 1/3 to 3/4 the length of the rotating growth vial.
[0120] In addition, the paddles may be modified to comprise
strainers or sieves for dissociating cell aggregates. That is, the
paddles may comprise pores that dissociate the cell aggregates,
where the pores range in size from 10 to 400 microns in size, or
from 20 to 200 microns in size, or from 30 to 100 microns in size.
In some embodiments of the automated instruments, there may be two
different types of rotating growth vials present, one type without
fins and/or strainers or sieves present for cell growth, and one
with fins or features and with strainers or sieves for cell
dissociation where cells and medium are transferred to and between
the growth vial and dissociation vial by an automated liquid
handling system.
[0121] FIG. 3C is a perspective view of one embodiment of a cell
growth device 330. FIG. 3D depicts a cut-away view of the cell
growth device 330 from FIG. 3C. In both figures, the rotating
growth vial 300 is seen positioned inside a main housing 336 with
the extended lip 302 of the rotating growth vial 300 extending
above the main housing 336. Additionally, end housings 352, a lower
housing 332 and flanges 334 are indicated in both figures. Flanges
334 are used to attach the cell growth device 330 to
heating/cooling means or other structure (not shown). FIG. 3D
depicts additional detail. In FIG. 3D, upper bearing 342 and lower
bearing 340 are shown positioned within main housing 336. Upper
bearing 342 and lower bearing 340 support the vertical load of
rotating growth vial 300. Lower housing 332 contains the drive
motor 338. The cell growth device 330 of FIG. 3C may comprise two
light paths; a primary light path 344, and a secondary light path
350. Light path 344 corresponds to light path 310 positioned in the
constricted portion of the tapered-to-constricted portion of the
rotating growth vial 300, and light path 350 corresponds to light
path 308 in the tapered portion of the tapered-to-constricted
portion of the rotating growth via 316. Light paths 310 and 308 are
not shown in FIG. 3D but may be seen in FIG. 3A. In addition to
light paths 344 and 340, there is an emission board 348 to
illuminate the light path(s), and detector board 346 to detect the
light after the light travels through the cell culture liquid in
the rotating growth vial 300.
[0122] Cell growth monitoring can be performed by imaging, for
example, by allowing the microcarriers to settle and imaging the
bottom of the rotating growth vial. Alternatively, an aliquot of
the culture is removed and run through a flow cell for imaging. In
yet another alternative, the cells may express a fluorescent
protein and fluorescence is measured. In yet another alternative,
the cell density may be measured by light absorbance at 250-350 nm
at light path 310.
[0123] The motor 328 engages with drive mechanism 312 and is used
to rotate the rotating growth vial 300. In some embodiments, motor
338 is a brushless DC type drive motor with built-in drive controls
that can be set to hold a constant revolution per minute (RPM)
between 0 and about 3000 RPM. Alternatively, other motor types such
as a stepper, servo, brushed DC, and the like can be used.
Optionally, the motor 338 may also have direction control to allow
reversing of the rotational direction, and a tachometer to sense
and report actual RPM. The motor is controlled by a processor (not
shown) according to, e.g., standard protocols programmed into the
processor and/or user input, and the motor may be configured to
vary RPM to cause axial precession of the cell culture thereby
enhancing mixing, e.g., to prevent cell aggregation, increase
aeration, and optimize cellular respiration.
[0124] Main housing 336, end housings 352 and lower housing 332 of
the cell growth device 330 may be fabricated from any suitable,
robust material including aluminum, stainless steel, or other
thermally conductive materials, including plastics. These
structures or portions thereof can be created through various
techniques, e.g., metal fabrication, injection molding, creation of
structural layers that are fused, etc. Whereas the rotating growth
vial 300 is envisioned in some embodiments to be reusable, but
preferably is consumable, the other components of the cell growth
device 330 are preferably reusable and function as a stand-alone
benchtop device or as a module in a multi-module cell processing
system.
[0125] FIG. 3E illustrates a cell growth device 330 as part of an
assembly comprising the cell growth device 330 of FIG. 3C coupled
to light source 390, detector 392, and thermal components 394. The
rotating growth vial 300 is inserted into the cell growth device.
Components of the light source 390 and detector 392 (e.g., such as
a photodiode with gain control to cover 5-log) are coupled to the
main housing of the cell growth device. The lower housing 332 that
houses the motor that rotates the rotating growth vial 300 is
illustrated, as is one of the flanges 334 that secures the cell
growth device 330 to the assembly. Also, the thermal components 394
illustrated are a Peltier device or thermoelectric cooler. In this
embodiment, thermal control is accomplished by attachment and
electrical integration of the cell growth device 330 to the thermal
components 394 via the flange 334 on the base of the lower housing
332. Thermoelectric coolers are capable of "pumping" heat to either
side of a junction, either cooling a surface or heating a surface
depending on the direction of current flow. In one embodiment, a
thermistor is used to measure the temperature of the main housing
and then, through a standard electronic
proportional-integral-derivative (PID) controller loop, the
rotating growth vial 300 is controlled to approximately
+/-0.5.degree. C. In yet another alternative, the detector is
replaced with an imaging camera. The geometry of the constricted
portion of the rotating growth vial 300 containing light path 310
is further tapered to collect settled cell aggregates or
microcarriers coated with cells when rotation is paused. The
stacked cell aggregates or microcarriers with cells are imaged.
Total cell number can be derived from the height of the stacked
cell aggregates. Total cell number can be derived from the combined
height of the microcarriers coated with cells and the observed
confluency of cells on a subset of microcarriers.
[0126] In use, cells are inoculated (cells can be pipetted, e.g.,
from an automated liquid handling system or by a user) into
pre-filled growth media of a rotating growth vial 300 by piercing
though the foil seal or film. The programmed software of the cell
growth device 330 sets the control temperature for growth,
typically 30.degree. C., then slowly starts the rotation of the
rotating growth vial 300. The cell/growth media mixture slowly
moves vertically up the wall due to centrifugal force allowing the
rotating growth vial 300 to expose a large surface area of the
mixture to an O.sub.2 or CO.sub.2 environment. If enhanced mixing
is required, e.g., to optimize growth conditions, the speed of the
vial rotation can be varied to cause an axial precession of the
liquid, and/or a complete directional change can be performed at
programmed intervals.
[0127] In addition to imaging, other cell growth parameters can be
measured. Other optional measures of cell growth may be made
including spectroscopy using visible, UV, or near infrared (NIR)
light, measuring, e.g., the concentration of nutrients and/or
wastes in the cell culture and/or other spectral properties can be
measured via, e.g., dielectric impedance spectroscopy, visible
fluorescence, fluorescence polarization, or luminescence.
Additionally, the cell growth device 330 may include additional
sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH,
conductivity, and the like. For additional details regarding
rotating growth vials and cell growth devices see U.S. Pat. Nos.
10,435,662; and 10,443,031; and U.S. Ser. No. 16/552,981, filed 7
Aug. 2019; and Ser. No. 16/780,640, filed 3 Feb. 2020.
Tangential Flow Filtration Module
[0128] An alternative to the rotating growth module for growing
cells off-instrument or as a growth module for an automated
instrument is a tangential flow filtration (TFF) module as shown in
FIGS. 4A-4G. The TFF module shown in FIGS. 4A-4G is a module that
can grow, perform buffer exchange, concentrate cells and dissociate
cells so that the cells may be transfected or transduced with the
nucleic acids needed for engineering or editing the cell's
genome.
[0129] FIG. 4A shows a retentate member 422 (top), permeate member
420 (middle) and a tangential flow assembly 410 (bottom) comprising
the retentate member 422, membrane 424 (not seen in FIG. 4A), and
permeate member 420 (also not seen). In FIG. 4A, retentate member
422 comprises a tangential flow channel 402, which has a serpentine
configuration that initiates at one lower corner of retentate
member 422--specifically at retentate port 428--traverses across
and up then down and across retentate member 422, ending in the
other lower corner of retentate member 422 at a second retentate
port 428. Also seen on retentate member 422 are energy directors
491, which circumscribe the region where a membrane or filter (not
seen in this FIG. 4A) is seated, as well as interdigitate between
areas of channel 402. Energy directors 491 in this embodiment mate
with and serve to facilitate ultrasonic welding or bonding of
retentate member 422 with permeate/filtrate member 420 via the
energy director component 491 on permeate/filtrate member 420 (at
right). Additionally, countersinks 423 can be seen, two on the
bottom one at the top middle of retentate member 422. Countersinks
423 are used to couple and tangential flow assembly 410 to a
reservoir assembly (not seen in this FIG. 4A but see FIG. 4B).
[0130] Permeate/filtrate member 420 is seen in the middle of FIG.
4A and comprises, in addition to energy director 491, through-holes
for retentate ports 428 at each bottom corner (which mate with the
through-holes for retentate ports 428 at the bottom corners of
retentate member 422), as well as a tangential flow channel 402 and
two permeate/filtrate ports 426 positioned at the top and center of
permeate member 420. The tangential flow channel 402 structure in
this embodiment has a serpentine configuration and an undulating
geometry, although other geometries may be used. Permeate member
420 also comprises countersinks 423, coincident with the
countersinks 423 on retentate member 420.
[0131] At bottom is a tangential flow assembly 410 comprising the
retentate member 422 and permeate member 420 seen in this FIG. 4A.
In this view, retentate member 422 is "on top" of the view, a
membrane (not seen in this view of the assembly) would be adjacent
and under retentate member 422 and permeate member 420 (also not
seen in this view of the assembly) is adjacent to and beneath the
membrane. Again countersinks 423 are seen, where the countersinks
in the retentate member 422 and the permeate member 420 are
coincident and configured to mate with threads or mating elements
for the countersinks disposed on a reservoir assembly (not seen in
FIG. 4A but see FIG. 4B).
[0132] A membrane or filter is disposed between the retentate and
permeate members, where fluids can flow through the membrane but
cells cannot and are thus retained in the flow channel disposed in
the retentate member. Filters or membranes appropriate for use in
the TFF device/module are those that are solvent resistant, are
contamination free during filtration, and are able to retain the
types and sizes of cells of interest. For example, in order to
retain small cell types, pore sizes can be as low as 0.5 .mu.m,
however for other cell types, the pore sizes can be as high as 20
.mu.m. Indeed, the pore sizes useful in the TFF device/module
include filters with sizes from 0.50 .mu.m and larger. The filters
may be fabricated from any suitable non-reactive material including
cellulose mixed ester (cellulose nitrate and acetate) (CME),
polycarbonate (PC), polyvinylidene fluoride (PVDF),
polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon,
glass fiber, or metal substrates as in the case of laser or
electrochemical etching.
[0133] The length of the channel structure 402 may vary depending
on the volume of the cell culture to be grown. The length of the
channel structure typically is from 60 mm to 300 mm, or from 70 mm
to 200 mm, or from 80 mm to 100 mm. The cross-section configuration
of the flow channel 402 may be round, elliptical, oval, square,
rectangular, trapezoidal, or irregular. If square, rectangular, or
another shape with generally straight sides, the cross section may
be from about 10 .mu.m to 1000 .mu.m wide, or from 200 .mu.m to 800
.mu.m wide, or from 300 .mu.m to 700 .mu.m wide, or from 400 .mu.m
to 600 .mu.m wide; and from about 10 .mu.m to 1000 .mu.m high, or
from 200 .mu.m to 800 .mu.m high, or from 300 .mu.m to 700 .mu.m
high, or from 400 .mu.m to 600 .mu.m high. If the cross section of
the flow channel 302 is generally round, oval or elliptical, the
radius of the channel may be from about 50 .mu.m to 1000 .mu.m in
hydraulic radius, or from 5 .mu.m to 800 .mu.m in hydraulic radius,
or from 200 .mu.m to 700 .mu.m in hydraulic radius, or from 300
.mu.m to 600 .mu.m wide in hydraulic radius, or from about 200 to
500 .mu.m in hydraulic radius. Moreover, the volume of the channel
in the retentate 422 and permeate 420 members may be different
depending on the depth of the channel in each member.
[0134] FIG. 4B shows front perspective (right) and rear perspective
(left) views of a reservoir assembly 450 configured to be used with
the tangential flow assembly 410 seen in FIG. 4A. Seen in the front
perspective view (e.g., "front" being the side of reservoir
assembly 450 that is coupled to the tangential flow assembly 410
seen in FIG. 4A) are retentate reservoirs 452 on either side of
permeate reservoir 454. Also seen are permeate ports 426, retentate
ports 428, and three threads or mating elements 425 for
countersinks 423 (countersinks 423 not seen in this FIG. 4B).
Threads or mating elements 425 for countersinks 423 are configured
to mate or couple the tangential flow assembly 410 (seen in FIG.
4A) to reservoir assembly 450. Alternatively, or in addition,
fasteners, sonic welding or heat stakes may be used to mate or
couple the tangential flow assembly 410 to reservoir assembly 450.
In addition is seen gasket 445 covering the top of reservoir
assembly 450. Gasket 445 is described in detail in relation to FIG.
4E. At left in FIG. 4B is a rear perspective view of reservoir
assembly 450, where "rear" is the side of reservoir assembly 450
that is not coupled to the tangential flow assembly. Seen are
retentate reservoirs 452, permeate reservoir 454, and gasket
445.
[0135] The TFF device may be fabricated from any robust material in
which channels (and channel branches) may be milled including
stainless steel, silicon, glass, aluminum, or plastics including
cyclic-olefin copolymer (COC), cyclo-olefin polymer (COP),
polystyrene, polyvinyl chloride, polyethylene, polyethylene,
polypropylene, acrylonitrile butadiene, polycarbonate,
polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),
polysulfone, and polyurethane, and co-polymers of these and other
polymers. If the TFF device/module is disposable, preferably it is
made of plastic. In some embodiments, the material used to
fabricate the TFF device/module is thermally-conductive so that the
cell culture may be heated or cooled to a desired temperature. In
certain embodiments, the TFF device is formed by precision
mechanical machining, laser machining, electro discharge machining
(for metal devices); wet or dry etching (for silicon devices); dry
or wet etching, powder or sandblasting, photostructuring (for glass
devices); or thermoforming, injection molding, hot embossing, or
laser machining (for plastic devices) using the materials mentioned
above that are amenable to this mass production techniques.
[0136] FIG. 4C depicts a top-down view of the reservoir assemblies
450 shown in FIG. 4B. FIG. 4D depicts a cover 444 for reservoir
assembly 450 shown in FIGS. 4B and 4E depicts a gasket 445 that in
operation is disposed on cover 444 of reservoir assemblies 450
shown in FIG. 4B. FIG. 4C is a top-down view of reservoir assembly
450, showing the tops of the two retentate reservoirs 452, one on
either side of permeate reservoir 454. Also seen are grooves 432
that will mate with a pneumatic port (not shown), and fluid
channels 434 that reside at the bottom of retentate reservoirs 452,
which fluidically couple the retentate reservoirs 452 with the
retentate ports 428 (not shown), via the through-holes for the
retentate ports in permeate member 420 and membrane 424 (also not
shown). FIG. 4D depicts a cover 444 that is configured to be
disposed upon the top of reservoir assembly 450. Cover 444 has
round cut-outs at the top of retentate reservoirs 452 and
permeate/filtrate reservoir 454. Again, at the bottom of retentate
reservoirs 452 fluid channels 434 can be seen, where fluid channels
434 fluidically couple retentate reservoirs 452 with the retentate
ports 428 (not shown). Also shown are three pneumatic ports 430 for
each retentate reservoir 452 and permeate/filtrate reservoir 454.
FIG. 4E depicts a gasket 445 that is configures to be disposed upon
the cover 444 of reservoir assembly 450. Seen are three fluid
transfer ports 442 for each retentate reservoir 452 and for
permeate/filtrate reservoir 454. Again, three pneumatic ports 330,
for each retentate reservoir 452 and for permeate/filtrate
reservoir 454, are shown.
[0137] The overall work flow for cell growth comprises loading a
cell culture to be grown into a first retentate reservoir,
preferably bubbling air or an appropriate gas through the cell
culture, passing or flowing the cell culture through the first
retentate port then tangentially through the TFF channel structure
while collecting medium or buffer through one or both of the
permeate ports 426, collecting the cell culture through a second
retentate port 428 into a second retentate reservoir, optionally
adding additional fresh or different medium to the cell culture and
optionally bubbling air or gas through the cell culture, then
repeating the process, all while measuring, e.g., the optical
density of the cell culture in the retentate reservoirs
continuously or at desired intervals. Again, cell growth monitoring
can be performed by imaging, for example, by allowing the
microcarriers to settle and imaging the bottom of the TFF retentate
reservoir. Alternatively, an aliquot of the culture is removed and
run through a flow cell for imaging. In yet another alternative,
the cells may express a fluorescent protein and fluorescence is
measured.
[0138] In the channel structure, the membrane bifurcating the flow
channels retains the cells on one side of the membrane (the
retentate side 422) and allows unwanted medium or buffer to flow
across the membrane into a filtrate or permeate side (e.g.,
permeate member 420) of the device. Bubbling air or other
appropriate gas through the cell culture both aerates and mixes the
culture to enhance cell growth. During the process, medium that is
removed during the flow through the channel structure is removed
through the permeate/filtrate ports 426. Alternatively, cells can
be grown in one reservoir with bubbling or agitation without
passing the cells through the TFF channel from one reservoir to the
other.
[0139] The overall work flow for cell concentration using the TFF
device/module involves flowing a cell culture or cell sample
tangentially through the channel structure. As with the cell growth
process, the membrane bifurcating the flow channels retains the
cells on one side of the membrane and allows unwanted medium or
buffer to flow across the membrane into a permeate/filtrate side
(e.g., permeate member 420) of the device. In this process, a fixed
volume of cells in medium or buffer is driven through the device
until the cell sample is collected into one of the retentate ports
428, and the medium/buffer that has passed through the membrane is
collected through one or both of the permeate/filtrate ports 426.
All types of prokaryotic and eukaryotic cells--both adherent and
non-adherent cells--can be grown in the TFF device. Adherent cells
may be grown on beads or other cell scaffolds suspended in medium
that flow through the TFF device.
[0140] The medium or buffer used to suspend the cells in the cell
concentration device/module may be any suitable medium or buffer
for the type of cells being transformed or transfected, such as
MEM, DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the
media may be provided in a reagent cartridge as part of a kit. For
culture of adherent cells, cells may be disposed on microcarriers
or other type of scaffold suspended in medium. The microcarriers of
particular use typically have a diameter of 50-500 .mu.m and have a
density slightly greater than that of the culture medium thus
facilitating an easy separation of cells and medium for, e.g.,
medium exchange yet the density must also be sufficiently low to
allow complete suspension of the carriers at a minimum stirring
rate in order to avoid hydrodynamic damage to the cells.
[0141] In both the cell growth and concentration processes, passing
the cell sample through the TFF device and collecting the cells in
one of the retentate ports 428 while collecting the medium in one
of the permeate/filtrate ports 426 is considered "one pass" of the
cell sample. The transfer between retentate reservoirs "flips" the
culture. The retentate and permeate ports collecting the cells and
medium, respectively, for a given pass reside on the same end of
TFF device/module with fluidic connections arranged so that there
are two distinct flow layers for the retentate and
permeate/filtrate sides, but if the retentate port 428 resides on
the retentate member of device/module (that is, the cells are
driven through the channel above the membrane and the filtrate
(medium) passes to the portion of the channel below the membrane),
the permeate/filtrate port 426 will reside on the permeate member
of device/module and vice versa (that is, if the cell sample is
driven through the channel below the membrane, the filtrate
(medium) passes to the portion of the channel above the membrane).
Due to the high pressures used to transfer the cell culture and
fluids through the flow channel of the TFF device, the effect of
gravity is negligible.
[0142] At the conclusion of a "pass" in either of the growth and
concentration processes, the cell sample is collected by passing
through the retentate port 428 and into the retentate reservoir
(not shown). To initiate another "pass", the cell sample is passed
again through the TFF device, this time in a flow direction that is
reversed from the first pass. The cell sample is collected by
passing through the retentate port 428 and into retentate reservoir
(not shown) on the opposite end of the device/module from the
retentate port 428 that was used to collect cells during the first
pass. Likewise, the medium/buffer that passes through the membrane
on the second pass is collected through the permeate port 426 on
the opposite end of the device/module from the permeate port 426
that was used to collect the filtrate during the first pass, or
through both ports. This alternating process of passing the
retentate (the concentrated cell sample) through the device/module
is repeated until the cells have been grown to a desired optical
density, and/or concentrated to a desired volume, and both permeate
ports (i.e., if there are more than one) can be open during the
passes to reduce operating time. In addition, buffer exchange may
be effected by adding a desired buffer (or fresh medium) to the
cell sample in the retentate reservoir, before initiating another
"pass", and repeating this process until the old medium or buffer
is diluted and filtered out and the cells reside in fresh medium or
buffer. Note that buffer exchange and cell growth may (and
typically do) take place simultaneously, and buffer exchange and
cell concentration may (and typically do) take place
simultaneously. For further information and alternative embodiments
on TFFs see, e.g., U.S. Ser. No. 16/798,302, filed 22 Feb.
2020.
[0143] In addition, the TFF may be modified to dissociate cells, as
shown in FIGS. 4F and 4G. That is, the TFF may be modified such
that the retentate reservoirs, in addition to being connected
through the flow channel that courses through the TFF device, are
connected directly by a conduit where the cells are passed from one
retentate reservoir to another without being sent through the flow
channel. In FIG. 4F, retentate reservoirs 452 are shown, connected
by conduit 462. In conduit 462 are placed one to many (e.g., in
FIG. 4G, there are three) strainers, frits or sieves 460 through
which aggregates of cells are passed to dissociate the aggregates.
As with the modified paddles or features in the rotating growth
vial, strainers, frits or sieves 460 comprise pores or openings
from 10 to 400 microns in size, or from 20 to 200 microns in size,
or from 30 to 100 microns in size configured to dissociate the cell
aggregates. That is, the TFF may be used to grow the cells--either
as aggregates or on microcarriers--passage the cells to increase
the number of cells, concentrate the cells and then finally the
cells may be routed through the direct conduit between the
retentate reservoirs to dissociate the cells for
transfection/transduction.
Bioreactor
[0144] In addition to the rotating growth vial module shown in
FIGS. 3A-3E and described in the related text, and the tangential
flow filtration module shown FIG. 4A-4G and described in the
related text, a bioreactor can be used to grow cells off-instrument
or to allow for cell growth and recovery on-instrument; e.g., as
one module of the multi-module automated instrument. Additionally,
the bioreactor can be used to generate emulsions using the impeller
and oil for encapsulated delivery of editing reagents, e.g., by
encapsulating cells and small reagent delivery vehicles in
dissolvable gel beads, or by encapsulating cells and larger
non-dissolvable reagent delivery vehicles to form uniform droplets
with a thin aqueous layer. The bioreactor may also be used to break
emulsions either those formed in the bioreactor itself or those
formed on a microfluidic device and transferred to the
bioreactor--using, e.g., the action of the impeller and a breaking
agent such as perfluoro-1-octanol. Further, the bioreactor supports
cell selection/enrichment, via expressed antibiotic markers in the
growth process or via expressed antibodies coupled to magnetic
beads and a magnet associated with the bioreactor. There are many
bioreactors known in the art, including those described in, e.g.,
WO 2019/046766; 10,699,519; 10,633,625; 10,577,576; 10,294,447;
10,240,117; 10,179,898; 10,370,629; and 9,175,259; and those
available from Lonza Group Ltd. (Basel, Switzerland); Miltenyi
Biotec (Bergisch Gladbach, Germany), Terumo BCT (Lakewood, Colo.)
and Sartorius GmbH (Gottingen, Germany).
[0145] FIG. 5A shows one embodiment of a bioreactor assembly 500
for cell growth in the automated multi-module cell processing
instruments described herein. Unlike most bioreactors that are used
to support fermentation or other processes with an eye to
harvesting the products produced by organisms grown in the
bioreactor, the present bioreactor (and the processes performed
therein) is configured to grow cells, monitor cell growth (e.g.,
via optical means and/or capacitance), passage cells, transfect or
transduce cells, select and/or enrich cells, and support editing,
expansion and harvesting of edited cells. Bioreactor assembly 500
comprises a growth vessel 501 comprising tapered a main body 504
with a lid assembly 502 comprising ports 508, including an optional
motor integration port 510 driving impeller 506 via impeller shaft
552. The tapered shape of main body 504 of the vessel 501 along
with, in some embodiments, dual impellers allows for working with a
larger dynamic range of volumes, such as, e.g., up to 500 ml and as
low as 100 ml for rapid sedimentation of the microcarriers. In
addition, the low volume is useful for magnetic bead separation or
enrichment as described above.
[0146] Bioreactor assembly 500 further comprises bioreactor stand
assembly 503 comprising a main body 512 and vessel holder 514
comprising a heat jacket or other heating means (not shown, but see
FIG. 5E) into which the main body 504 of vessel 501 is disposed in
operation. The main body 504 of vessel 501 is biocompatible and
preferably transparent--in some embodiments, in the UV and IR range
as well as the visible spectrum--so that the growing cells can be
visualized by, e.g., cameras or sensors integrated into lid
assembly 502 or through viewing apertures or slots in the main body
512 of bioreactor stand assembly 503 (not shown in this FIG. 5A,
but see FIG. 5E).
[0147] Bioreactor assembly 500 supports growth of cells from a
500,000 cell input to a 10 billion cell output, or from a 1 million
cell input to a 25 billion cell output, or from a 5 million cell
input to a 50 billion cell output or combinations of these ranges
depending on, e.g., the size of main body 504 of vessel 501, the
medium used to grow the cells, whether the cells are adherent or
non-adherent. The bioreactor that comprises assembly 500 supports
growth of both adherent and non-adherent cells, wherein adherent
cells are typically grown of microcarriers as described in detail
above and supra or as spheroids. Alternatively, another option for
growing mammalian cells in the bioreactor described herein is
growing single cells in suspension using a specialized medium such
as that developed by ACCELLTA.TM. (Haifa, Israel). As described
above, cells grown in this medium must be adapted to this process
over many cell passages; however, once adapted the cells can be
grown to a density of >40 million cells/ml and expanded
50-100.times. in approximately a week, depending on cell type.
[0148] Main body 504 of vessel 501 preferably is manufactured by
injection molding, as is, in some embodiments, impeller 506 and the
impeller shaft (not shown). Impeller 506 also may be fabricated
from stainless steel, metal, plastics or the polymers listed infra.
Injection molding allows for flexibility in size and configuration
and also allows for, e.g., volume markings to be added to the main
body 504 of vessel 501. Additionally, material from which the main
body 504 of vessel 501 is fabricated should be able to be cooled to
about 4.degree. C. or lower and heated to about 55.degree. C. or
higher to accommodate cell growth. Further, the material that is
used to fabricate the vial preferably is able to withstand
temperatures up to 55.degree. C. without deformation. Suitable
materials for main body 504 of vessel 501 include those described
for the rotating growth vial described in relation to FIGS. 3A and
3B and the TFF device described in relation to FIG. 4A-4E,
including cyclic olefin copolymer (COC), glass, polyvinyl chloride,
polyethylene, polyetheretherketone (PEEK), polypropylene,
polycarbonate, poly(methyl methacrylate) (PMMA), polysulfone,
poly(dimethylsiloxane), cyclo-olefin polymer (COP), and co-polymers
of these and other polymers. Preferred materials include
polypropylene, polycarbonate, or polystyrene. The material used for
fabrication may depend on the cell type to be grown, and is
conducive to growth of both adherent and non-adherent cells. The
main body 504 of vessel 501 may be reusable or, alternatively, may
be manufactured and configured for a single use. In one embodiment,
main body 504 of vessel 501 may support cell culture volumes of 25
ml to 500 ml, but may be scaled up to support cell culture volumes
of up to 3 L.
[0149] The bioreactor stand assembly comprises a stand or frame
550, a main body 512 which holds the vessel 501 during operation.
The stand/frame 550 and main body 512 are fabricated from stainless
steel, other metals, or polymer/plastics. The bioreactor main body
further comprises a heat jacket (not seen in FIG. 5A, but see FIG.
5E) to maintain the bioreactor main body 504--and thus the cell
culture--at a desired temperature. Essentially, the stand assembly
can host a set of sensors and cameras to monitor cell culture.
[0150] FIG. 5B depicts a top-down view of one embodiment of vessel
lid assembly 502. Vessel lid assembly 502 is configured to be
air-tight, providing a sealed, sterile environment for cell growth
as well as to provide biosafety thus maintaining a closed system.
Vessel lid assembly 502 and the main body 504 of vessel 501 can be
sealed via fasteners such as screws, using biocompatible glues, or
the two components may be ultrasonically welded. Vessel lid
assembly 502 is some embodiments is fabricated from stainless steel
such as S316L stainless steel but may also be fabricated from
metals, other polymers (such as those listed supra) or plastics. As
seen in this FIG. 5B--as well as in FIG. 5A--vessel lid assembly
502 comprises a number of different ports to accommodate liquid
addition and removal; gas addition and removal; for insertion of
sensors to monitor culture parameters (described in more detail
infra); to accommodate one or more cameras or other optical
sensors; to provide access to the main body 504 of vessel 501 by,
e.g., a liquid handling device; and to accommodate a motor for
motor integration to drive one or more impellers 506. Exemplary
ports depicted in FIG. 5B include three liquid-in ports 516 (at 4
o'clock, 6 o'clock and 8 o'clock), one liquid-out port 522 (at 11
'clock), a capacitance sensor 518 (at 9 o'clock), one "gas in" port
524 (at 12 o'clock), one "gas out" port 520 (at 10 o'clock), an
optical sensor 526 (at 1 o'clock), a rupture disc 528 at 2 o'clock,
a self-sealing port 530 (at 3 o'clock) to provide access to the
main body 504 of growth vessel 501; and (a temperature probe 532
(at 5 o'clock).
[0151] The ports 508 shown in vessel lid assembly 502 in this FIG.
5B are exemplary only and it should be apparent to one of ordinary
skill in the art given the present disclosure that, e.g., a single
liquid-in port 516 could be used to accommodate addition of all
liquids to the cell culture rather than having a liquid-in port for
each different liquid added to the cell culture. Similarly, there
may be more than one gas-in port 524, such as one for each gas,
e.g., O.sub.2, CO.sub.2 that may be added. In addition, although a
temperature probe 532 is shown, a temperature probe alternatively
may be located on the outside of vessel holder 514 of bioreactor
stand assembly 503 separate from or integrated into heater jacket
548 (not seen in this FIG. 5B, but see FIG. 5E). A self-sealing
port 530, if present, allows access to the main body 504 of vessel
501 for, e.g., a pipette, syringe, or other liquid delivery system
via a gantry (not shown). As shown in FIG. 5A, additionally there
may be a motor integration port to drive the impeller(s), although
in other configurations of vessel 501 may alternatively integrate
the motor drive at the bottom of the main body 504 of vessel 501.
Vessel lid assembly 502 may also comprise a camera port for viewing
and monitoring the cells.
[0152] Additional sensors include those that detect O.sub.2
concentration, a CO.sub.2 concentration, culture pH, lactate
concentration, glucose concentration, biomass, and optical density.
The sensors may use optical (e.g., fluorescence detection),
electrochemical, or capacitance sensing and either be reusable or
configured and fabricated for single-use. Sensors appropriate for
use in the bioreactor are available from Omega Engineering (Norwalk
Conn.); PreSens Precision Sensing (Regensburg, Germany); C-CIT
Sensors AG (Waedenswil, Switzerland), and ABER Instruments Ltd.
(Alexandria, Va.). In one embodiment, optical density is measured
using a reflective optical density sensor to facilitate
sterilization, improve dynamic range and simplify mechanical
assembly. The rupture disc, if present, provides safety in a
pressurized environment, and is programmed to rupture if a
threshold pressure is exceeded in the bioreactor. If the cell
culture in the bioreactor vessel is a culture of adherent cells,
microcarriers may be used as described supra. In such an instance,
the liquid-out port may comprise a filter such as a stainless steel
or plastic (e.g., polyvinylidene difluoride (PVDF), nylon,
polypropylene, polybutylene, acetal, polyethylene, or polyamide)
filter or frit to prevent microcarriers from being drawn out of the
culture during, e.g., medium exchange, but to allow dead cells to
be withdrawn from the vessel. The microcarriers used for initial
cell growth can be nanoporous (where pore sizes are typically
<20 nm in size), microporous (with pores between >20 nm to
<1 .mu.m in size), or macroporous (with pores between >1
.mu.m in size, e.g., 20 .mu.m) and the microcarriers are typically
50-200 .mu.m in diameter; thus the pore size of the filter or frit
in the liquid-out port will differ depending on microcarrier
size.
[0153] The microcarriers used for cell growth depend on cell type
and desired cell numbers, and typically include a coating of a
natural or synthetic extracellular matrix or cell adhesion
promoters (e.g., antibodies to cell surface proteins or
poly-L-lysine) to promote cell growth and adherence. Microcarriers
for cell culture are widely commercially available from, e.g.,
Millipore Sigma, (St. Louis, Mo., USA); ThermoFisher Scientific
(Waltham, Mass., USA); Pall Corp. (Port Washington, N.Y., USA); GE
Life Sciences (Marlborough, Mass., USA); and Corning Life Sciences
(Tewkesbury, Mass., USA). As for the extracellular matrix, natural
matrices include collagen, fibrin and vitronectin (available, e.g.,
from ESBio, Alameda, Calif., USA), and synthetic matrices include
MATRIGEL.RTM. (Corning Life Sciences, Tewkesbury, Mass., USA),
GELTREX.TM. (ThermoFisher Scientific, Waltham, Mass., USA),
CULTREX.RTM. (Trevigen, Gaithersburg, Md., USA), biomemetic
hydrogels available from Cellendes (Tubingen, Germany); and
tissue-specific extracellular matrices available from Xylyx
(Brooklyn, N.Y., USA); further, denovoMatrix (Dresden, Germany)
offers screenMATRIX.TM., a tool that facilitates rapid testing of a
large variety of cell microenvironments (e.g., extracellular
matrices) for optimizing growth of the cells of interest.
[0154] FIG. 5C is a side view of the main body 504 of vessel 501. A
portion of vessel lid assembly 502 can be seen, as well as two
impellers 506a and 506b. Also seen are a lactate/glucose sensor
probe 534, a pH, O.sub.2, CO.sub.2 sensor 536 (such as a
PRESENS.TM. integrated optical sensor (Precision Sensing GmbH,
(Regensburg, Germany)), and a viable biomass sensor 538 (such as,
e.g., the FUTURA PICO.TM. capacitance sensor (ABER, Alexandria
Va.)). In some embodiments, flat regions are fabricated onto the
main body 504 of vessel 501 to reduce optical loss, simplify spot
placement and simplify fluorescent measurement of pH, dO.sub.2, and
dCO.sub.2.
[0155] FIG. 5D shows exemplary design guidelines for a one-impeller
embodiment (left) and a two-impeller embodiment (right) of the main
body 504 of vessel 501, including four exemplary impeller
configurations. The embodiment of the INSCRIPTA.TM. bioreactor
vessel 501 main body 504 as shown in this FIG. 5D has a total
volume of 820 ml and supports culture volumes from 25 ml to 500 ml.
As mentioned above, the impellers (and impeller shaft) may be
injection molded or may be fabricated from stainless steel, other
biocompatible metals, polymers or plastics and preferably comprised
polished surfaces to facilitate sterilization. The impeller may be
configured as a turbine-, pitched-blade-, hydrofoil- or marine-type
impeller. In a two-impeller configuration, the impellers may be of
the same type or different types. In the bioreactors described
herein (the "INSCRIPTA.TM. bioreactors") and used to generated the
data in Examples IV-XI, agitation is provided at 0-100 rpm, or
40-80 rpm, or approximately 70 rpm during cell growth (depending on
the cell type being cultured); however, lower or higher revolutions
per minute may be used depending on the volume of the main body 504
of vessel 501, the type of cells being cultured, whether the cells
are adherent and being grown on microcarriers or the cells are
non-adherent, and the size and configuration of the impellers. The
impeller may turn in a clockwise direction, a counter-clockwise
direction or the impeller may change direction (oscillate) or stop
at desired intervals, particularly during cell detachment from the
microcarriers. Also, intermittent agitation may be applied, e.g.,
agitating for 10 minutes every 30 minutes, or agitating for 1
minute every 5 minutes or any other desired pattern. Additionally,
impeller rpm is often increased (e.g., up to 4000 rpm) when the
cells are being detached from microcarriers. Although the present
embodiment of INSCRIPTA.TM. bioreactor utilizes one or more
impellers for cell growth, alternative embodiments of the
INSCRIPTA.TM. bioreactor described herein may utilize bubbling or
other physical mixing means.
[0156] Also seen in FIG. 5D is an equation that gives a range for
exemplary bioreactor dimensions base on the height (H) and
thickness (T) of the main body of vessel 504. For example,
D=0.25-05*T means the impeller diameter could be one quarter or one
half of the main body of vessel 504 thickness, T. C is the
clearance of the impeller from the bottom of the main body of
vessel 504, which can be 0.15 to 0.5 times the thickness. It should
be apparent to one of ordinary skill in the art given the present
disclosure that these numbers are just one embodiment and the
ranges may be larger. The bioreactor vessel 501 main body 504
comprises an 8-10 mm clearance from the bottom of the main body 504
of vessel 501 to the lower impeller 506b and the lower impeller
506b and the upper impeller 506a are approximately 40 mm apart.
[0157] FIG. 5E is a side view of the vessel holder portion 514 of
the bioreactor stand main body 512 of the bioreactor stand assembly
503. Inner surface 540 of vessel holder 514 is indicated and shown
are camera or fiber optic ports 546 for monitoring, e.g., cell
growth and viability; O.sub.2 and CO.sub.2 levels, and pH. The
vessel holder portion 514 of the bioreactor stand main body 512 may
also provide illumination using LED lights, such as a ring of LED
lights (not shown). FIG. 5F is a side perspective view of the
assembled bioreactor without sensors 542. Seen are vessel lid
assembly 502, bioreactor stand assembly 503, bioreactor stand main
body 512 into which the main body 504 of vessel 501 (not seen in
FIG. 5E) is inserted. FIG. 5G is a lower side perspective view of
bioreactor assembly 500 showing bioreactor stand assembly 503,
bioreactor stand main body 512, vessel lid assembly 502 and two
camera mounts 544. Surrounding bioreactor stand main body 512 is
heater jacket 548.
[0158] FIG. 5H is an exemplary diagram of the bioreactor fluidics.
Fluidics and pneumatics are designed to establish a cell culture
environment conducive for mammalian cell growth, including iPSCs.
Fluidic circuits are designed to deliver and/or remove cell medium,
buffers, microcarriers and additional reagents needed for growth,
maintenance, selection and passaging of the cells in the automated
closed culture instrument. The pneumatic circuits are designed to
deliver the appropriate gas mixture and humidity for the chosen
cell type, and may comprise line-in filters to prevent any
contaminants from reaching the bioreactor.
[0159] FIG. 5I is a block diagram for an exemplary bioreactor
control system. The control system is designed to control and
automate the fluidics, pneumatics and sensor function in a closed
system and without human intervention. In one embodiment, the
control system is based on state-machines with a user editable
state order and parameters using Json and jsonette config files.
State-machines allow for dynamic control of several aspects of the
bioreactor with a single computer.
[0160] In use, the bioreactor described herein is used for cell
growth and expansion--either before or after the cells are
transfected in droplets--as well as for medium exchange and cell
concentration. Also mentioned supra, the bioreactor may be used to
generate emulsions/droplets using the impeller and added oil, e.g.,
by encapsulating cells and small reagent delivery vehicles in
dissolvable gel beads, or by encapsulating cells and larger
non-dissolvable reagent delivery vehicles to form uniform droplets
with a thin aqueous layer. The bioreactor may also be used to break
emulsions--either those formed in the bioreactor itself or those
formed on a microfluidic device and transferred to the
bioreactor--using, e.g., the action of the impeller and a breaking
agent such as perfluoro-1-octanol.
[0161] Medium/buffer exchange is in one embodiment accomplished
using gravitational sedimentation and aspiration via a filter in
the liquid-out port where the filter is of an appropriate size to
retain microcarriers (see, e.g., Example VII, infra). In one
embodiment used with the present bioreactor, a frit with pore size
100 .mu.m was used and microcarriers with diameters or 120-225
.mu.m were used in the cell culture. Sedimentation was accomplished
in approximately 2-3 minutes for a 100 ml culture and 4-5 minutes
for a 500 ml culture. The medium was aspirated at >100 ml/min
rate. In addition to clearing the medium from the main body 504 of
vessel 501, dead cells were removed as well. If sedimentation is
used, the microcarriers do not typically accumulate on the filter;
however, if accumulation is detected, the medium in the liquid-out
port can be pushed back into main body 504 of vessel 501 in a
pulse. In some embodiments--particularly those where sedimentation
is not used--a cycle of aspiration, release (push back), aspiration
and release (push back) may be performed. Experimental results show
that medium exchange (aspiration) at .about.200 ml/min does not
impact cell growth (see FIG. 14).
Exemplary Solid Wall Module for Delivering Reagent Bundle Droplets
to Cells
[0162] Returning to the solid wall delivery embodiment, FIGS. 6A-6D
depict a solid wall device 650 for partitioning mammalian cells in
microwells in a solid wall isolation, incubation, and normalization
(SWIIN) module. FIG. 6A depicts an embodiment of a SWIIN module 650
from an exploded top perspective view. In SWIIN module 650 the
retentate member is formed on the bottom of a top of a SWIIN module
component and the permeate member is formed on the top of the
bottom of a SWIIN module component.
[0163] The SWIIN module 650 in FIG. 6A comprises from the top down,
a reservoir gasket or cover 658, a retentate member 604 (where a
retentate flow channel cannot be seen in this FIG. 6A), a
perforated member 601 swaged with a filter (filter not seen in FIG.
6A), a permeate member 608 comprising integrated reservoirs
(permeate reservoirs 652 and retentate reservoirs 654, one of each
seen in this perspective view), and two reservoir seals 662, which
seal the bottom of permeate reservoirs 652 and retentate reservoirs
654. A permeate channel 660a can be seen disposed on the top of
permeate member 608, defined by a raised portion 676 of serpentine
channel 660a, and ultrasonic tabs 664 can be seen disposed on the
top of permeate member 608 as well. The perforations that form the
wells on perforated member 601 are not seen in this FIG. 6A;
however, through-holes 666 to accommodate the ultrasonic tabs 664
are seen. In addition, supports 670 are disposed at either end of
SWIIN module 650 to support SWIIN module 650 and to elevate
permeate member 608 and retentate member 604 above reservoirs 652
and 654 to minimize bubbles or air entering the fluid path from the
permeate reservoir to serpentine channel 660a or the fluid path
from the retentate reservoir to serpentine channel 660b (neither
fluid path is seen in this FIG. 6A). In yet another alternative,
the filter is bonded to perforated member 601 using an adhesive. In
yet another alternative, the filter bottom of each well is
patterned with two electrodes to perform electroporation. These
electrodes may be patterned in an interdigitated format with one
electrode being the anode and the other electrode being the
cathode. In yet another alternative, one electrode is in the
permeate chamber and the other electrode is in the retentate
chamber. In an alternative approach, reagents for delivery are
printed on the filter in the wells during the manufacturing
process. Printing reagents on the filter could be performed before
or after swaging or bonding filter membrane to the perforated
metal.
[0164] In this FIG. 6A, it can be seen that the serpentine channel
660a that is disposed on the top of permeate member 608 traverses
permeate member 608 for most of the length of permeate member
608--except for the portion of permeate member 608 that comprises
permeate reservoirs 652 and retentate reservoirs 654--and for most
of the width of permeate member 608. As used herein with respect to
the distribution channels in the retentate member or permeate
member, "most of the length" means about 95% of the length of the
retentate member or permeate member, or about 90%, 85%, 80%, 75%,
or 70% of the length of the retentate member or permeate member. As
used herein with respect to the distribution channels in the
retentate member or permeate member, "most of the width" means
about 95% of the width of the retentate member or permeate member,
or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate
member or permeate member.
[0165] In this embodiment of a SWIIN module, the perforated member
includes through-holes to accommodate ultrasonic tabs disposed on
the permeate member. Thus, in this embodiment the perforated member
is fabricated from 316 stainless steel, and the perforations form
the walls of microwells while a membrane is used to form the bottom
of the microwells. Typically, the perforations (microwells) are
approximately 150 .mu.m-200 .mu.m in diameter, and the perforated
member is approximately 125 .mu.m deep, resulting in microwells
having a volume of approximately 2.5 nL, with a total of
approximately 200,000 microwells, although volumes as small as 0.1
nL may be used. The distance between the microwells is
approximately 279 .mu.m center-to-center. Though here the
microwells have a volume of approximately 2.5 nl, depending on the
mammalian cell type being edited the volume of the microwells may
be from 5 to 100 nl, or preferably from 10 to 50 nl, and even more
preferably from 10 to 25 nl. As for the membrane, membranes
appropriate for use are solvent resistant, contamination free, and
are able to retain the types and sizes of cells of interest. For
example, in order to retain mammalian cells, the pore sizes can be
as high as 10.0 .mu.m-20.0 .mu.m or more. Indeed, the pore sizes
useful in the cell concentration device/module include filters with
sizes from 0.10 .mu.m, 0.11 .mu.m, 0.12 .mu.m, 0.13 .mu.m, 0.14
.mu.m, 0.15 .mu.m, 0.16 .mu.m, 0.17 .mu.m, 0.18 .mu.m, 0.19 .mu.m,
0.20 .mu.m, 0.21 .mu.m, 0.22 .mu.m, 0.23 .mu.m, 0.24 .mu.m, 0.25
.mu.m, 0.26 .mu.m, 0.27 .mu.m, 0.28 .mu.m, 0.29 .mu.m, 0.30 .mu.m,
0.31 .mu.m, 0.32 .mu.m, 0.33 .mu.m, 0.34 .mu.m, 0.35 .mu.m, 0.36
.mu.m, 0.37 .mu.m, 0.38 .mu.m, 0.39 .mu.m, 0.40 .mu.m, 0.41 .mu.m,
0.42 .mu.m, 0.43 .mu.m, 0.44 .mu.m, 0.45 .mu.m, 0.46 .mu.m, 0.47
.mu.m, 0.48 .mu.m, 0.49 .mu.m, 0.50 .mu.m and larger. The filters
may be fabricated from any suitable material including cellulose
mixed ester (cellulose nitrate and acetate) (CME), polycarbonate
(PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES),
polytetrafluoroethylene (PTFE), nylon, or glass fiber.
[0166] The cross-section configuration of the mated serpentine
channel may be round, elliptical, oval, square, rectangular,
trapezoidal, or irregular. If square, rectangular, or another shape
with generally straight sides, the cross section may be from about
2 mm to 15 mm wide, or from 3 mm to 12 mm wide, or from 5 mm to 10
mm wide. If the cross section of the mated serpentine channel is
generally round, oval or elliptical, the radius of the channel may
be from about 3 mm to 20 mm in hydraulic radius, or from 5 mm to 15
mm in hydraulic radius, or from 8 mm to 12 mm in hydraulic
radius.
[0167] Serpentine channels 660a and 660b can have approximately the
same volume or a different volume. For example, each "side" or
portion 660a, 660b of the serpentine channel may have a volume of,
e.g., 2 mL, or serpentine channel 660a of permeate member 608 may
have a volume of 2 mL, and the serpentine channel 660b of retentate
member 604 may have a volume of, e.g., 3 mL. The volume of fluid in
the serpentine channel may range from about 2 mL to about 80 mL, or
about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL
(note these volumes apply to a SWIIN module comprising a, e.g.,
50-500K perforation member). The volume of the reservoirs may range
from 5 mL to 50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or
from 10 mL to 20 mL, and the volumes of all reservoirs may be the
same or the volumes of the reservoirs may differ (e.g., the volume
of the permeate reservoirs is greater than that of the retentate
reservoirs).
[0168] The serpentine channel portions 660a and 660b of the
permeate member 608 and retentate member 604, respectively, are
approximately 200 mm long, 130 mm wide, and 4 mm thick, though in
other embodiments, the retentate and permeate members can be from
75 mm to 400 mm in length, or from 100 mm to 300 mm in length, or
from 150 mm to 250 mm in length; from 50 mm to 250 mm in width, or
from 75 mm to 200 mm in width, or from 100 mm to 150 mm in width;
and from 2 mm to 15 mm in thickness, or from 4 mm to 10 mm in
thickness, or from 5 mm to 8 mm in thickness. In some embodiments
the retentate (and permeate) members may be fabricated from PMMA
(poly(methyl methacrylate)) or other materials may be used,
including polycarbonate, cyclic olefin co-polymer (COC), glass,
polyvinyl chloride, polyethylene, polyetheretherketone (PEEK),
poly(dimethylsiloxane), polypropylene, polysulfone, polyurethane,
and co-polymers of these and other polymers. Preferably at least
the retentate member is fabricated from a transparent material so
that the cells can be visualized. For example, a video camera may
be used to monitor cell growth by, e.g., density change
measurements based on an image of an empty well, with phase
contrast, or if, e.g., a chromogenic marker, such as a chromogenic
protein, is used to add a distinguishable color to the cells.
Chromogenic markers such as blitzen blue, dreidel teal, virginia
violet, vixen purple, prancer purple, tinsel purple, maccabee
purple, donner magenta, cupid pink, seraphina pink, scrooge orange,
and leor orange (the Chromogenic Protein Paintbox, all available
from ATUM (Newark, Calif.)) obviate the need to use fluorescence,
although fluorescent cell markers, fluorescent proteins, and
chemiluminescent cell markers may also be used.
[0169] Because the retentate member preferably is transparent,
colony growth in the SWIIN module can be monitored by automated
devices such as the growth monitor sold by IncuCyte (Ann Arbor,
Mich.) (see also, Choudhry, PLos One, 11(2):e0148469 (2016)).
[0170] Due to the heating and cooling of the SWIIN module,
condensation may accumulate on the retentate member which may
interfere with accurate visualization of the growing cell colonies.
Condensation of the SWIIN module 650 may be controlled by, e.g.,
moving heated air over the top of (e.g., retentate member) of the
SWIIN module 650, or by applying a transparent heated lid over at
least the serpentine channel portion 660b of the retentate member
604 (not shown).
[0171] In SWIIN module 650 cells and medium are flowed into
serpentine channel 560b from ports in retentate member 604, and the
cells settle in the microwells while the medium passes through the
membrane into serpentine channel 660a in permeate member 608. The
cells are retained in the microwells of perforated member 601 as
the cells cannot travel through membrane 603. Appropriate medium
may be introduced into permeate member 608 through permeate ports
611. The medium flows upward through membrane 603 to nourish the
cells in the microwells (perforations) of perforated member 601.
Additionally, buffer exchange can be effected by cycling medium
through the retentate and permeate members.
[0172] Once editing has taken place, the cells may be flushed from
the microwells by applying fluid or air pressure (or both) to the
permeate member serpentine channel 660a and thus to filter 603 and
pooled. Alternatively, the cell growth medium may be removed and
trypsin, collagen or pronase applied until the cells are
dislodged.
[0173] FIG. 6B is a top perspective view of a SWIIN module with the
retentate and perforated members in partial cross section. In this
FIG. 6B, it can be seen that serpentine channel 660a is disposed on
the top of permeate member 608, is defined by raised portions 676
and traverses permeate member 608 for most of the length and width
of permeate member 608 except for the portion of permeate member
608 that comprises the permeate and retentate reservoirs (note only
one retentate reservoir 652 can be seen in this FIG. 6B). Moving
from left to right, reservoir gasket 658 is disposed upon the
integrated reservoir cover 678 (cover not seen in this FIG. 6B) of
retentate member 604. Gasket 658 comprises reservoir access
apertures 632a, 632b, 632c, and 632d, as well as pneumatic ports
633a, 633b, 633c and 633d. Also, at the far-left end is support
670. Disposed under permeate reservoir 652 can be seen one of two
reservoir seals 662. In addition to the retentate member being in
cross section, the perforated member 601 and filter 603 (filter 603
is not seen in this FIG. 6B) are in cross section. Note that there
are a number of ultrasonic tabs 664 disposed at the right end of
SWIIN module 650 and on raised portion 676 which defines the
channel turns of serpentine channel 660a, including ultrasonic tabs
664 extending through through-holes 666 of perforated member 601.
There is also a support 670 at the end distal reservoirs 652, 654
of permeate member 608.
[0174] FIG. 6C is a side perspective view of an assembled SWIIIN
module 650, including, from right to left, reservoir gasket 658
disposed upon integrated reservoir cover 678 (not seen) of
retentate member 604. Gasket 658 may be fabricated from rubber,
silicone, nitrile rubber, polytetrafluoroethylene, a plastic
polymer such as polychlorotrifluoroethylene, or other flexible,
compressible material. Gasket 658 comprises reservoir access
apertures 632a, 632b, 632c, and 632d, as well as pneumatic ports
633a, 633b, 633c and 633d. Also at the far-left end is support 670
of permeate member 608. In addition, permeate reservoir 652 can be
seen, as well as one reservoir seal 662. At the far-right end is a
second support 670.
[0175] Imaging of cell colonies growing in the wells of the SWIIN
is desired in most implementations for, e.g., monitoring both cell
growth and device performance and imaging is necessary for
cherry-picking implementations. Real-time monitoring of cell growth
in the SWIIN requires backlighting, retentate plate (top plate)
condensation management and a system-level approach to temperature
control, air flow, and thermal management. In some implementations,
imaging employs a camera or CCD device with sufficient resolution
to be able to image individual wells. For example, in some
configurations a camera with a 9-pixel pitch is used (that is,
there are 9 pixels center-to-center for each well). Processing the
images may, in some implementations, utilize reading the images in
grayscale, rating each pixel from low to high, where wells with no
cells will be brightest (due to full or nearly-full light
transmission from the backlight) and wells with cells will be dim
(due to cells blocking light transmission from the backlight).
After processing the images, thresholding is performed to determine
which pixels will be called "bright" or "dim", spot finding is
performed to find bright pixels and arrange them into blocks, and
then the spots are arranged on a hexagonal grid of pixels that
correspond to the spots. Once arranged, the measure of intensity of
each well is extracted, by, e.g., looking at one or more pixels in
the middle of the spot, looking at several to many pixels at random
or pre-set positions, or averaging X number of pixels in the spot.
In addition, background intensity may be subtracted. Thresholding
is again used to call each well positive (e.g., containing cells)
or negative (e.g., no cells in the well). The imaging information
may be used in several ways, including taking images at time points
for monitoring cell growth and to assure proper fluid flow in the
serpentine channel 660.
[0176] FIG. 6D depicts a side view of the embodiment of the SWIIN
module in FIGS. 6A-6C further comprising a heat management system
including a heater and a heated cover. The heater cover facilitates
the condensation management that is required for imaging. Assembly
698 comprises a SWIIN module 650 seen lengthwise in cross section,
where one permeate reservoir 652 is seen. Disposed immediately upon
SWIIN module 650 is cover 694 and disposed immediately below SWIIN
module 650 is backlight 680, which allows for imaging. Beneath and
adjacent to the backlight and SWIIN module is insulation 682, which
is disposed over a heatsink 684. In this FIG. 6D, the fins of the
heatsink would be in-out of the page. In addition, there is also
axial fan 686 and heat sink 688, as well as two thermoelectric
coolers 692, and a controller 690 to control the pneumatics,
thermoelectric coolers, fan, solenoid valves, etc. The arrows
denote cool air coming into the unit and hot air being removed from
the unit. It should be noted that control of heating allows for
growth of many different types of cells as well as cells that are,
e.g., temperature sensitive, etc. Temperature control allows for
protocols to be adjusted to account for differences in
transformation efficiency, cell growth and viability. For more
details regarding solid wall isolation incubation and normalization
devices see U.S. Pat. No. 10,533,152; 10,550,363; 10,633,626;
10,633,627; 10,647,958; 10,760,043; 10,723,995; 10,844,344,
10,801,008; and 10,851,339. For alternative isolation, incubation
and normalization modules, see U.S. Pat. Nos. 10,532,324;
10,625,212, 10,744,462; 10,752,874; and 10,835,869.
[0177] In addition, the SWIIN described may be adapted to
dissociate cells in a manner similar to that of the TFF. That is,
the SWIIN may be modified such that the retentate reservoirs, in
addition to being connected through the flow channel that courses
through the SWIIN device, are connected directly by a conduit where
the cells are passed from one retentate reservoir to another
without being sent through the flow channel. See, e.g., FIGS. 4F
and 4G.
EXAMPLES
[0178] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention, nor are they intended to represent or imply that
the experiments below are all of or the only experiments performed.
It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as
shown in the specific aspects without departing from the spirit or
scope of the invention as broadly described. The present aspects
are, therefore, to be considered in all respects as illustrative
and not restrictive.
Example I: Efficiency of Transfection, Bulk Vs. Droplets
[0179] To test the efficiency of transformation of mammalian cells
in droplets, HEK293T cells were transfected with a plasmid driving
expression of a fluorescent reporter molecule (dsRed). For the
transfection, lipoplex solutions were prepared:
TABLE-US-00001 uL 20 rxns Tube 1: Optimem 25 500 L3000 1.5 30 Tube
2: Optimem 25 500 DNA (980 ng/uL) 1.02 20.4 P3000 2 40
[0180] To assemble the lipoplexes, 400 .mu.L from each tube was
loaded into separate syringes and were mixed and emulsified in a
CHIPSHOP.TM. microfluidic droplet generator chip 162 (Jeno,
Germany). The flow rates were as follows: aqueous, 500 .mu.L per
hour; hydrofluoroether (e.g., carrier phase)+fluoro-surfactant, 1
mL per hour. The droplets were incubated for 10 minutes and then
demulsified with 20% perfluorooctanol in hydrofluoroether.
[0181] For the bulk transfection, 100 .mu.L of the lipoplex
solutions from each tube were mixed and 50 .mu.L of drop-assembled
or bulk-assembled lipoplex mixture to each well of previously
seeded 100 K HEK293T cells in 0.5 mL complete medium.
[0182] For the in-droplet transfection, 500 .mu.L of drop-assembled
lipoplex mixture was loaded in one syringe and 500 .mu.L of the
cell suspension (at a concentration of 4M cells/mL) was loaded into
a second syringe. The two mixtures were emulsified and loaded
separately onto a CHIPSHOP.TM. microfluidic droplet generator chip
163 (Jeno, Germany) and incubated at 37.degree. C. for 30 minutes.
The flow rates were as follows: aqueous phases (lipoplex and
cells), 1 mL per hour; hydrofluoroether (e.g., carrier
phase)+fluoro-surfactant, 2 mL per hour. The aqueous phases were
demulsified with 20% perfluorooctanol in hydrofluoroether and the
sample was diluted 1:10 in complete medium. The sample was then
split into two tubes with one sample being washed in complete
medium. The samples were then plated. FIG. 7A depicts the lipoplex
assembly and transfection processes.
[0183] Forty-eight hours after plating, cells were subjected to
fluorescence-based sorting using a FACSMelody.TM. Cell Sorter
(Becton Dickenson, Franklin Lakes, N.J.) based on dsRed reporter
expression levels. The results are shown in FIG. 7B. Cells
transfected with either the bulk method or the droplet method were
transfected with similar efficiency (>60%) as reported by
percent dsRed-positive cells.
Example II: Demonstration of Droplet Partitioning
[0184] An experiment was performed to test the integrity of cells
in droplet partitions. HEK293T cells were transfected in droplets
as described in Example II above in two separate transformations:
one transformation was with a plasmid (EEV-CAG-BFP) driving
expression of blue fluorescent protein and one transformation was
with a plasmid (EEV-CAG-dsRed) driving expression of red
fluorescent protein. The cells were then demulsified and pooled as
shown in FIG. 8A. After pooling, the cells were split into two
aliquots, with one aliquot washed one time and the other aliquot
washed three times. The cells were analyzed by flow cytometry for
expression of the fluorescent reporter proteins and the results are
shown in FIG. 8B. Note that there was very little cross
contamination between BFP+ and dsRed+ cells; that is, there was a
low percentage (<3%) of BFP+ and dsRed+ cells.
[0185] FIG. 9A is a graph plotting the predicted yield of
differently-sized amplified nucleic acids (e.g., 500 bp, 2000 bp
and 10,000 bp) per droplet in differently-sized droplets. FIG. 9B
is a graph plotting the cell coverage at 3e6 copies/cell for 500 bp
amplicons for droplets of differing size.
Example III: GFP to BFP Conversion Assay
[0186] A GFP to BFP reporter cell line was created using mammalian
cells with a stably integrated genomic copy of the GFP gene
(HEK293T-GFP). These cell lines enabled phenotypic detection of
genomic edits by various different mechanisms, including flow
cytometry, fluorescent cell imaging, and genotypic detection by
sequencing of the genome-integrated GFP gene. Lack of editing or
perfect repair of cut events in the GFP gene result in cells that
remain GFP-positive. Cut events that are repaired by the
Non-Homologous End-Joining (NHEJ) pathway often result in
nucleotide insertion or deletion events (indels), resulting in
frame-shift mutations in the coding sequence that cause loss of GFP
gene expression and fluorescence. Cut events that are repaired by
the Homology-Directed Repair (HDR) pathway, using the GFP to BFP
HDR donor as a repair template, result in conversion of the cell
fluorescence profile from that of GFP to that of BFP.
[0187] FIG. 10 is a simplified depiction of an engine vector
(described below) and amplified editing cassettes. The editing
cassettes were designed that are complementary to regions proximal
to the EGFP-to-BFP editing site. FIG. 11 shows an exemplary scheme
for amplifying the editing cassettes in droplets. This scheme
allows for the addition of a promoter, in this case the U6
promoter, to the editing cassette to produce an expression-ready
cfgRNA construct. The cfgRNA cassettes comprised, from 5' to 3', a
first priming site (P1), a gRNA spacer region (SR), a gRNA scaffold
region, a donor DNA or homology arm (HA) comprising both a silent
PAM mutation (SPM) and a target site mutation (TSM), and a second
priming site (P2). A first primer construct comprising the U6
promoter with a region complementary to the first priming site (P1)
and a second primer complementary to the second priming site were
used to amplify the cfgRNA cassette, resulting in an expression
cassette comprising from 5' to 3', the U6 promoter, the first
priming site (P1), the gRNA spacer region (SR), the gRNA scaffold
region, the donor DNA or homology arm (HA) comprising both the
silent PAM mutation (SPM) and the target site mutation (TSM), and
the second priming site (P2). In addition to the U6 promoter, the
U6 primer may comprise other functional or non-functional groups
(here, denoted by "R") such as a phosphate group, an amine group, a
biotin tag, a barcode and/or an NLS peptide.
Example IV: Biocompatibility of Bioreactor Materials
[0188] Biocompatibility of bioreactor relevant materials were
screened in plate cultures using conditioned media. mTeSR.TM.Plus
serum-free, feeder-free cell culture medium (STEMCELL Technologies
Canada INC., Vancouver, BC) was incubated with the material of
interest (i.e., stainless steel and polycarbonate) for at least 72
hours at 4.degree. C. for conditioning the cell culture media.
WTC11 iPSCs were seeded on 6-well plates and conditioned media was
used to grow cells in standard incubators at 37.degree. C., 5%
CO.sub.2 and >95% relative humidity. Control cultures were grown
similarly to the tested conditions except the medium was not
conditioned with any materials and the medium was kept at 4.degree.
C. for 72 hours before the start of cultures.
[0189] Cells were seeded on Matrigel coated 6-well plates
(CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates (Corning,
Inc., Glendale, Ariz.)) and cultured with their respective
conditioned (tested sample) or unconditioned media (control) and
CloneR.TM. (STEMCELL Technologies Canada INC., Vancouver, BC) for
the first 24 hours. After the first 24 hours, cell media was
exchanged with fresh conditioned (tested sample) or unconditioned
media (control) without CloneR, and maintained up to 72 hours where
cells reached confluency. Cell counts and viabilities were assessed
at 12-hours, 36-hour and 60-hour time points after lifting cells
from the Matrigel CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well
plates (Corning, Inc., Glendale, Ariz.) plates using RelesR.TM.
reagent (following the manufacturer's instructions) (STEMCELL
Technologies Canada INC., Vancouver, BC) and the cells were
quantified on a NucleoCounter NC-200 (Chemometec, Allerod, Denmark)
automated cell counting instrument following the manufacturer's
instructions.
[0190] FIGS. 12A and 12B show the results of these experiments.
FIGS. 12A and 12B demonstrate neither growth nor viability is
impacted by the choice of materials for fabrication of the main
body 504 of vessel 501 (polycarbonate), vessel lid assembly 502
(stainless steel), impeller 506 (stainless steel or polycarbonate),
or medium exchange frit (stainless steel). All components were
sterilized before conditioning.
Example V: Optimal Working Volume
[0191] The bioreactor described herein was tested for optimal
working volume. For sensor operation, minimum optimal volume was
set to 100 ml with sensor clearance at 10 mm from the bottom of the
main body of the vessel. 10 million WTC11 iPSCs were seeded on 0.5
g of 10m/ml laminin L-521 coated Enhanced Attachment microcarriers
(Corning, Inc., Glendale, Ariz.) in 40 ml and 100 ml mTeSR.TM.Plus
serum-free, feeder-free cell culture medium (STEMCELL Technologies
Canada INC., Vancouver, BC) and CloneR (STEMCELL Technologies
Canada INC., Vancouver, BC in CORNING.RTM. spinner flasks (Corning,
Inc., Glendale, Ariz.). Impeller agitation was set to 70 rpm using
a CHIMAREC.TM. direct stirrer (ThermoFisher Scientific, Waltham
Mass.). A first media exchange was performed at 24 hours, and then
at every 48th hour with fresh mTeSR.TM.Plus serum-free, feeder-free
cell culture medium (STEMCELL Technologies Canada INC., Vancouver,
BC) (no CloneR). The cells attached to the microcarriers were
quantified at 12-hour and 36-hour time points on a NucleoCounter
NC-200 (Chemometec, Allerod, Denmark) automated cell counting
instrument following the manufacturer's instructions. Cell counts
indicated similar cell seeding efficiencies at 40 ml and 100 ml
seeding volumes (data not shown).
Example VI: Assessing Growth in Bioreactor to Traditional Plating
and Spinner Flask Culture
[0192] Experiments were performed to assess whether cell growth in
the INSCRIPTA.TM. bioreactor described herein is equivalent to
traditional plate and spinner flask culture conditions. Ten million
WTC11 iPSCs were seeded on 0.5 g of 10 .mu.g/ml laminin L-521
coated Enhanced Attachment microcarriers (Corning, Inc., Glendale,
Ariz.) in 100 ml mTeSR.TM.Plus serum-free, feeder-free cell culture
medium (STEMCELL Technologies Canada INC., Vancouver, BC) and
CloneR (STEMCELL Technologies Canada INC., Vancouver, BC) in the
INSCRIPTA.TM. bioreactor and in CORNING.RTM. spinner flasks
(Corning, Inc., Glendale, Ariz.). Impeller agitation was performed
at 70 rpm for both the INSCRIPTA.TM. bioreactor and CORNING.RTM.
spinners. A control culture was also seeded on Matrigel coated
6-well plates (CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates
(Corning, Inc., Glendale, Ariz.)) using 500 k cells per one well.
The cells were maintained at 37.degree. C., 5% CO.sub.2 and >95%
relative humidity throughout the culture period. The first media
exchange was performed at 24 hours, and then at every 48th hour
with fresh mTeSR.TM.Plus serum-free, feeder-free cell culture
medium (STEMCELL Technologies Canada INC., Vancouver, BC) (no
CloneR) using 100 ml for microcarrier cultures and 2 ml per well
for 6-well plates. Cell counts were quantified at 12-hour, 36-hour
and 60-hour time points on a NucleoCounter NC-200 (Chemometec,
Allerod, Denmark) automated cell counting instrument following the
manufacturer's instructions.
[0193] The results are shown in FIG. 13. The graph at top shows
similar numbers of iPSC cells at 10, 20, 30, 40, 50, 60, and 70
hours after seeding. The graph at bottom left shows similar results
were obtained for iPSC cell expansion in three different
INSCRIPTA.TM. bioreactors. The graph at bottom right shows the
results obtained for iPSC cell expansion in four different
CORNING.RTM. spinner flasks. Growth curves plotted using these cell
counts indicated similar cell growth curves under the conditions
tested. The 6-well plate control counts were scaled assuming an
initial cell seeding number of 10 million cells for comparison.
Additional INSCRIPTA.TM. bioreactors and CORNING.RTM. spinner
flasks were seeded on different days using the same methods to
compare cell growth curve variations and showed similar variation
across INSCRIPTA.TM. bioreactors and CORNING.RTM. spinners.
Example VII: Effect of Medium Exchange
[0194] Ten million WTC11 iPSCs were seeded on 0.5 g of 10 .mu.g/ml
laminin L-521 coated Enhanced Attachment microcarriers (Corning,
Inc., Glendale, Ariz.) in 100 ml mTeSR.TM.Plus serum-free,
feeder-free cell culture medium (STEMCELL Technologies Canada INC.,
Vancouver, BC) and CloneR (STEMCELL Technologies Canada INC.,
Vancouver, BC) in INSCRIPTA.TM. bioreactors and CORNING.RTM.
spinner flasks. Impeller agitation was performed at 70 rpm for both
the INSCRIPTA.TM. bioreactors and the CORNING.RTM. spinners. A
6-well plate control culture was also seeded on CORNING.RTM.
BIOCOAT.TM. MATRIGEL.RTM. 6-well plates (Corning, Inc., Glendale,
Ariz.) was also seeded using 500 k cells per one well. The cells
were maintained at 37.degree. C., 5% CO.sub.2 and >95% relative
humidity throughout the culture period. A first media exchange was
performed at 24 hours, and then at every 48th hour with fresh
mTeSR.TM.Plus serum-free, feeder-free cell culture medium (STEMCELL
Technologies Canada INC., Vancouver, BC) (no CloneR) using 100 ml
for microcarrier cultures and 2 ml per well for 6-well plates.
Media exchanges on the INSCRIPTA.TM. bioreactors were performed
using a frit system as follows: Impeller agitation was stopped and
the microcarriers were allowed to settle gravitationally for 5
minutes. After settling, >90% of the spent media was aspirated
from the INSCRIPTA.TM. bioreactor through a frit connected to a
peristaltic pump operating at 200 ml/min flow rate. The frit
consisted of .about.100 micron pores while the microcarriers ranged
from 120-225 micron in diameter. As such, microcarriers were
retained in the bioreactor but spent media and dead cells were
aspirated out of the bioreactor vessel. As a comparison, media
exchange in CORNING.RTM. spinner flasks and 6-well plates were
performed using a serological pipette connected to an aspirator
(BVC Professional Aspiration System (Vacuubrand, Essex Conn.)). In
all conditions, fresh media was added manually using a serological
pipette. Cell counts were quantified at 20-hour, 44-hour and
68-hour time points on a NucleoCounter NC-200 (Chemometec, Allerod,
Denmark) automated cell counting instrument following the
manufacturer's instructions. The results of four replicates in the
INSCRIPTA.TM. bioreactors are shown in FIG. 14. Growth curves
plotted using these cell counts indicated that the media exchange
approach through a frit does not have any noticeable impact on cell
growth. The 6-well plate control counts were scaled assuming an
initial cell seeding number of 10 million cells for comparison.
During the process there was no accumulation of microcarriers on
the frit in the liquid-out port.
Example VIII: Effect of Impeller Shear on Cell Viability and
Reproducibility
[0195] Cell detachment from microcarriers may be achieved using an
impeller agitation-based approach as follows: 10M cells were seeded
on 0.5 g of 10 .mu.g/ml laminin L-521 coated microcarriers
(Corning, Inc., Glendale, Ariz.), and expanded in the INSCRIPTA.TM.
bioreactor at 100 ml mTeSR.TM.Plus serum-free, feeder-free cell
culture medium (STEMCELL Technologies Canada INC., Vancouver, BC)
at 37.degree. C., 5% CO.sub.2, and >95% relative humidity as
described above. Once the cells reached >50 million cells as
determined by cell counting, the microcarriers were allowed to
settle gravitationally for 5 minutes, and >90% of the spent
media was aspirated. 100 ml phosphate buffered saline (PBS) was
added to microcarriers for washing and aspirated after 5 minutes.
100 ml RelesR (STEMCELL Technologies Canada INC., Vancouver, BC)
was added to the microcarriers and incubated at 37.degree. C. for 6
minutes. After 6 minutes, >90% of the RelesR (STEMCELL
Technologies Canada INC., Vancouver, BC) was aspirated and 100 ml
of cell media was added to the microcarriers to quench any
RelesR.
[0196] At this stage, impeller agitation was performed by rotating
the impeller at 2700 rpm in the clockwise direction for 15 seconds
first, and then at 2700 rpm in the counter-clockwise direction for
15 seconds. This bi-directional agitation for a total of 30 seconds
duration was defined as "one round" or "one cycle". Up to five
rounds/cycles of impeller agitation was tested in terms of cell
detachment efficiency. After detachment, the cell and microcarrier
suspension was transferred to a conical vessel. Cells and
microcarriers were separated using gravitational settling where the
microcarriers settle faster than the cells due to their larger
diameter. In another approach, the cell and microcarrier suspension
was passed through a strainer with 100 micron mesh size (e.g.,
CORNING.RTM. Sterile Cell strainer-100 micron, Corning, Inc.,
Glendale, Ariz.) to separate the cells from the microcarriers. As
control, a 1 ml aliquot of microcarrier culture was detached using
a P1000 pipette (PIPETMAN.RTM.) by passing the microcarriers
through the pipette 5 times. After detachment, post detachment
viability and the number of detached cells were quantified for
assessing detachment efficiency.
[0197] The results are shown in FIG. 15. The graph at top left of
FIG. 15 shows the percent post-detachment of the cells. The graph
top right in FIG. 15 shows the number of viable cells/ml
(.times.10.sup.5) out of .about.0.6M attached cells. The graph at
bottom left in FIG. 15 shows the number of cells/ml attached out of
.about.500K seeded. Finally, the graph at bottom right in FIG. 15
shows the attached fraction of cells after each cycle. Note that
viability remained around 90% after all of the first, third and
fifth cycles. The cells were effectively detached from the
microcarriers using the impeller agitation approach and showed
>90% post-detachment viability after up to 5 rounds of impeller
agitation, which was similar to the control. The re-seeding
efficiency of cells detached with impeller agitation were also
similar to the control case where .gtoreq.70% of the detached cells
were able to re-seed.
[0198] Reproducibility of impeller agitation-based passaging was
tested. Ten million cells were seeded on 0.5 g of 10m/ml laminin
L-521 coated microcarriers (Corning, Inc., Glendale, Ariz.), and
expanded in the INSCRIPTA.TM. bioreactor in 100 ml mTeSR.TM.Plus
serum-free, feeder-free cell culture medium (STEMCELL Technologies
Canada INC., Vancouver, BC) at 37.degree. C., 5% CO.sub.2, and
>95% relative humidity as described above. Once the cells
reached >50 million cells as determined by cell counting, the
microcarriers were allowed to settle gravitationally for 5 minutes
and >90% spent media was aspirated. 100 ml phosphate buffered
saline (PBS) was added to the microcarriers for washing and was
aspirated after 5 minutes. 100 ml RelesR (STEMCELL Technologies
Canada INC., Vancouver, BC) were added to the microcarriers and
incubated at 37.degree. C. for 6 minutes. After 6 minutes, >90%
of the RelesR was aspirated and 100 ml of cell media was added to
the microcarriers to quench any RelesR. At this stage impeller
agitation was performed by rotating the impeller at 2700 rpm in
clockwise direction for 15 seconds first, and then at 2700 rpm in
counter-clockwise direction for 15 seconds. This bi-directional
agitation for a total of 30 seconds duration was defined as "one
round" or "one cycle". Three rounds/cycles of impeller agitation
were used to detach the cells from microcarriers. After detachment,
the cell and microcarrier suspension was transferred to a conical
vessel. The cells and the microcarriers were separated using
gravitational settling where the microcarriers settle faster than
cells due to their larger diameter. Detached cells were re-seeded
on fresh microcarriers at 10 million cells per 0.5 g of
CORNING.RTM. laminin coated microcarriers (Corning, Inc., Glendale,
Ariz.), and re-seeding efficiencies were determined based on cell
counts at 24 hours after seeding. Passaging and re-seeding
efficiencies are quantified and shown in the FIG. 16. FIG. 16 at
top shows a simplified workflow for this process, as well as at
middle a table showing the efficiency of each step, and at bottom a
bar graph of passaging statistics for the indicated steps. The
results indicate that impeller-based passaging is reproducible and
allows for re-seeding of 30-65% of cells that were on the
microcarriers prior to detachment.
Example IX: Cell Re-Seeding and Expansion after Impeller
Passaging
[0199] Cell seeding and expansion after impeller passaging was
tested. Ten million WTC11 cells were seeded on 0.5 g of 10 .mu.g/ml
laminin L-521 coated microcarriers (Corning, Inc., Glendale,
Ariz.), and expanded in the INSCRIPTA.TM. bioreactor in 100 ml
mTeSR.TM.Plus serum-free, feeder-free cell culture medium (STEMCELL
Technologies Canada INC., Vancouver, BC) at 37.degree. C., 5%
CO.sub.2, and >95% relative humidity as described above. Once
the cells reached >50 million cells as determined by cell
counting, the impeller passaging protocol was implemented as
described above. After detachment, 10M detached cells were
re-seeded on 0.5 g of fresh laminin coated microcarriers (Corning,
Inc., Glendale, Ariz.) and expanded as described above. As a
control, an INSCRIPTA.TM. bioreactor was seeded with cells detached
from T75 flasks detached using standard protocols. Cell counts were
quantified at 20-hour, 44-hour and 68-hour time points on a
NucleoCounter NC-200 (Chemometec, Allerod, Denmark) automated cell
counting instrument following the manufacturer's instructions. The
results are shown in FIG. 17. FIG. 17 is a graph of triplicate
results demonstrating that cell seeding and expansion are
unaffected by impeller-shear passaging.
Example X: Ability of Cells to Maintain Stemness
[0200] The ability of the iPSCs to retain stemness during culture
and passaging was tested. Ten million cells were seeded on 0.5 g of
10 .mu.g/ml laminin L-521 coated microcarriers (Corning, Inc.,
Glendale, Ariz.), and expanded in an INSCRIPTA.TM. bioreactor in
mTeSR.TM.Plus serum-free, feeder-free cell culture medium (STEMCELL
Technologies Canada INC., Vancouver, BC) at 37.degree. C., 5%
CO.sub.2, and >95% relative humidity as described above. Once
the cells reached >50 million cells as determined by cell
counting, the impeller passaging protocol was implemented and 10M
detached cells were re-seeded onto fresh 0.5 g laminin coated
microcarriers (Corning, Inc., Glendale, Ariz.). This process was
repeated two more times and the cells were stained after final
detachment using antibodies (BIOLEGEND.RTM., San Diego Calif.)
specific to three stemness expression markers (TRA-1-60, OCT-3/4
and SOX-2) following the manufacturer's instructions, followed by
analysis using flow cytometry (BD FACSMelody.TM.) (Becton
Dickinson, Inc., Franklin Lakes, N.J.). Cells grown and impeller
passaged on the INSCRIPTA.TM. bioreactors showed expression of
stemness markers similar to the cells grown on Matrigel
(CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates (Corning,
Inc., Glendale, Ariz.)) and laminin coated plates (CORNING.RTM.
BIOCOAT.TM. laminin plates (Corning, Inc., Glendale, Ariz.)).
[0201] Stemness antibody staining was performed in the following
manner, with the equipment and materials listed in Table 1:
TABLE-US-00002 TABLE 1 Foxp3/Transcription Factor
Fixation/Permeabilization Concentrate and Diluent, ThermoFisher
Scientific, cat. # 00-5521-00 eBioscience .TM. Flow Cytometry
Staining Buffer, ThermoFisher Scientific, cat. # 00-4222-26
Anti-SOX2 (Brilliant Violet 421): Biolegend, cat. # 656114
Anti-OCT3/4 (Alexa488): Biolegend, cat. # 653706 Anti-TRA-1-60
(PE-Cy7): Biolegend, cat. # 330620 Anti-CD44 (PE-Cy5): ThermoFisher
Scientific, cat. # 15-0441-82 Anti-CD13 (PE-Cy7): Biolegend, cat. #
301712 Anti-NESTIN (Alexa488): Biolegend, cat. # 656812 Anti-SSEA4
(V450): BD Biosciences, cat. # 561156 FACSMelody .TM. flow
cytometer (Becton Dickinson, Inc., Franklin Lakes, NJ)
[0202] In a first step, a single-cell suspension was prepared and
centrifuged 5 minutes at 200.times.g. The cells were then washed in
an appropriate volume of DPBS and centrifuged again for 5 minutes
at 200.times.g. The supernatant was discarded and the pellet was
vortexed to dissociate the pellet. Fresh Foxp3
fixation/permeabilization working solution (ThermoFisher
Scientific, Waltham, Mass.) was prepared by mixing one part Foxp3
fixation/permeabilization concentrate with three parts Foxp3
fixation/permeabilization diluent and 1 ml was added to each tube
and each tube was then vortexed. The vortexed cells and
fixation/permeabilization working solution were incubated for 30-60
minutes in the dark at room temperature. A 1.times. working
solution of permeabilization buffer was prepared by mixing 1 part
10.times. permeabilization buffer with 9 parts dH.sub.2O and 2 ml
was added to each sample. The cells were centrifuged at
400-600.times.g for 5 minutes at room temperature and the
supernatant was discarded. The cell pellet was resuspended in
1.times. permeabilization buffer for a total volume of
approximately 100 .mu.l. The cells were diluted so that there were
no more than 10,000 cells/.mu.l, and 1M cells were transferred to a
fresh tube. The appropriate amount of directly-conjugated antibody
was dispensed into each tube. The cells were incubated for >30
minutes in the dark at room temperature. Two ml of 1.times.
permeabilization buffer was added to each tube and the samples were
centrifuged at 400-600.times.g for 5 minutes at room temperature
and the supernatant was discarded. The stained cells were suspended
in flow cytometry staining buffer.
[0203] The results are shown in FIG. 18. FIG. 18 at top are
histograms showing the fluorescent expression distribution measured
via flow cytometry of the cell population for individual sternness
marker expression. The x-axis shows the fluorescence signal and the
y-axis shows cell count. BR1 indicates results for INSCRIPTA.TM.
bioreactor 1, BR2 indicates results for INSCRIPTA.TM. bioreactor 2
(replicate), L1 indicates CORNING.RTM. BIOCOAT.TM. laminin plates
(Corning, Inc., Glendale, Ariz.), L2 indicates CORNING.RTM.
BIOCOAT.TM. laminin plates (Corning, Inc., Glendale, Ariz.)
(replicate), M1 indicates CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM.
6-well plates (Corning, Inc., Glendale, Ariz.), and M2 indicates
CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates (Corning,
Inc., Glendale, Ariz.) (replicate). A dark control was used for
comparison where the cells in one well from the M1 6-well plate are
prepared as the experimental cells but were not stained with
antibodies. Looking at the graph at bottom left of FIG. 18, note
that the percent of cells positive for the TRA-1-60 and SOX2 cell
surface markers was similar across culture conditions. Cell surface
marker OCT3/4 was a little lower (94-96%) in the cells grown in the
INSCRIPTA.TM. bioreactors than in the laminin plates (98%) and in
the MATRIGEL.RTM. plates (98%). The graph at right of FIG. 18 shows
the median fluorescence obtained for each of TRA 1-60, OCT3/4 and
SOX2 markers for each bioreactor, laminin plate and MATRIGEL.RTM.
plate replicate.
Example XI: Ability of Cells to Maintain Differentiation
Potential
[0204] To test whether cells grown in the INSCRIPTA.TM. bioreactor
would retain differentiation potential, ten million cells were
seeded on 0.5 g of 10 .mu.g/ml laminin L-521 coated microcarriers
(Corning, Inc., Glendale, Ariz.), and expanded in INSCRIPTA.TM.
Bioreactor in 100 ml mTeSR.TM.Plus serum-free, feeder-free cell
culture medium (STEMCELL Technologies Canada Inc., Vancouver, BC)
at 37.degree. C., 5% CO.sub.2, and >95% relative humidity as
described above. Once the cells reached >50 million cells as
determined by cell counting, the impeller passaging protocol as
described above in Example VII was implemented and 10M detached
cells were re-seeded onto 0.5 g fresh laminin coated microcarriers.
This process was repeated two more times, and after the final
detachment the cells were seeded on 12-well plates for trilineage
differentiation using a commercial protocol (STEMDIFF.TM. Trilinage
Differentiation Kit, STEMCELL Technologies Canada Inc., Vancouver,
BC). After trilineage differentiation, the cells from each lineage
were stained with antibodies specific to markers specific to that
lineage (available from BIOLEGEND.RTM., San Diego Calif. and
Miltenyi Biotec, San Diego, Calif.) following the manufacturer's
instructions. The cells grown and impeller-passaged on the
INSCRIPTA.TM. bioreactors showed expression of lineage-specific
markers similar to the cells grown on Matrigel and laminin coated
plates.
[0205] The tri-lineage differentiation antibody staining protocol
was performed in the following manner, with the equipment listed in
Table 2 and the antibodies listed in Table 3:
TABLE-US-00003 TABLE 2 Foxp3/Transcription Factor
Fixation/Permeabilization Concentrate and Diluent, ThermoFisher
Scientific, cat. # 00-5521-00 eBioscience .TM. Flow Cytometry
Staining Buffer, ThermoFisher Scientific, cat. # 00-4222-26 FACS
staining buffer (2% FBS, 1 mM EDTA, 0.5% BSA) FACS buffer (2% FBS,
1 mM EDTA) FACSMelody .TM. flow cytometer (Becton Dickinson, Inc.,
Franklin Lakes, NJ)
TABLE-US-00004 TABLE 3 Cell Antibody Marker Type Link Catalog #
Conjugate Isotype Isotype Conc. CXCR4 Mesoderm BioLegend 306518
BV421 Mouse BioLegend 1:200 IgG2a, .kappa. NCAM1 Mesoderm BioLegend
362510 PE-Cy7 Mouse BioLegend 1:200 IgG1, .kappa. Brachyury
Mesoderm SantaCruz sc- AF488 Mouse BioLegend 1:25 374321 IgG2b,
.kappa. AF488 Nestin Ectoderm BioLegend 656808 BV421 Mouse
BioLegend 1:400 IgG2a, .kappa. Otx-2 Ectoderm Miltenyi 130-121- Vio
B515 recombinant Miltenyi 1:100 202 hs IgG1 PAX6 Ectoderm Miltenyi
130-123- PE recombinant Miltenyi 1:400 250 hs IgG1 CXCR4 Endoderm
BioLegend 306518 BV421 Mouse BioLegend 1:400 IgG2a, .kappa. SOX17
Endoderm Miltenyi 130-111- Vio B515 recombinant Miltenyi 1:600 147
hs IgG1 FOXA2 Endoderm BD 561589 PE Mouse BD >1:20 Biosciences
IgG1, .kappa. Biosciences
[0206] A single-cell suspension was prepared by lifting cells with
TrypLE.TM. SELECT (ThermoFisher Scientific, Waltham, Mass., USA)
and was centrifuged for 5 minutes at 200.times.g. The cells were
washed in DPBS and centrifuged a second time. The cells were fixed
with a Foxp3 kit (ThermoFisher Scientific, Waltam, Mass.) according
to the manufacturer's instructions. Following incubation at room
temperature in the dark for 30-60 minutes, 1 ml Foxp3
fixation/permeabilization working solution was added. Each sample
contained .ltoreq.10M cells. A 1.times. working solution of
permeabilization buffer was prepared by mixing 1 part of 10.times.
Permeabilization Buffer with 9 parts of distilled water and 2 ml of
1x permeabilization buffer was added to each tube. The samples were
centrifuged at 400-600.times.g for 5 minutes at room temperature.
The supernatant was discarded and the pellet was resuspended in
residual volume of 1.times. permeabilization buffer for a total
volume of approximately 100 .mu.l. The cells were diluted so that
there were no more than 10,000 cells/.mu.l in a 96-well V- or
U-bottom plate. A master mix of antibodies per cell lineage in FACS
staining buffer was prepared. Approximately 500,000 cells were
stained in 50 .mu.l of staining solution. The cells were incubated
on ice in the dark for at least 30 minutes. 150 .mu.l of FACS
buffer was added to each well. The cells were then centrifuged at
500.times.g for 5 minutes at room temperature and the supernatant
was discarded. The cells were resuspended in FACS buffer and
analyzed by a flow cytometer on the FACSMelody.TM. flow
cytometer.
[0207] The results are shown in FIG. 19A-19F. FIGS. 19A, 19C and
19E comprise bar graphs showing % positive cells for endoderm
markers CXCR4 and SOX17; mesoderm markers NCAM1 and CXCR4; and
ectoderm markers NESTIN, OTX2 and PAX6. FIGS. 19B, 19D and 19E
comprise bar graphs showing median fluorescence obtained for the
endoderm, mesoderm and ectoderm markers. BR1 indicates results for
INSCRIPTA.TM. bioreactor 1, BR2 indicates results for INSCRIPTA.TM.
bioreactor 2 (replicate), L1 indicates CORNING.RTM. BIOCOAT.TM.
laminin plates (Corning, Inc., Glendale, Ariz.), L2 indicates
CORNING.RTM. BIOCOAT.TM. laminin plates (Corning, Inc., Glendale,
Ariz.) (replicate), M1 indicates CORNING.RTM. BIOCOAT.TM.
MATRIGEL.RTM. 6-well plates (Corning, Inc., Glendale, Ariz.), and
M2 indicates CORNING.RTM. BIOCOAT.TM. MATRIGEL.RTM. 6-well plates
(Corning, Inc., Glendale, Ariz.) (replicate). Note that the cells
grown in the bioreactors maintain differentiation potential roughly
equivalent to cells grown in the laminin plates and MATRIGEL.RTM.
plates. A pluripotent control was used, where the pluripotent
control were cells that were not differentiated using the STEMDIFF
medium (STEMDIFF.TM. Trilinage Differentiation Kit, STEMCELL
Technologies Canada Inc., Vancouver, BC) but were maintained in
mTeSRPlus medium (STEMCELL Technologies Canada INC., Vancouver,
BC).
[0208] While this invention is satisfied by embodiments in many
different forms, as described in detail in connection with
preferred embodiments of the invention, it is understood that the
present disclosure is to be considered as exemplary of the
principles of the invention and is not intended to limit the
invention to the specific embodiments illustrated and described
herein. Numerous variations may be made by persons skilled in the
art without departure from the spirit of the invention. The scope
of the invention will be measured by the appended claims and their
equivalents. The abstract and the title are snot to be construed as
limiting the scope of the present invention, as their purpose is to
enable the appropriate authorities, as well as the general public,
to quickly determine the general nature of the invention. In the
claims that follow, unless the term "means" is used, none of the
features or elements recited therein should be construed as
means-plus-function limitations pursuant to 35 U.S.C. .sctn. 112,
6.
* * * * *