U.S. patent application number 15/734128 was filed with the patent office on 2022-09-15 for method and apparatus for processing tissue samples.
The applicant listed for this patent is S2 Genomics, Inc.. Invention is credited to John Bashkin, Kaiwan Chear, David Eberhart, Stevan Bogdan Jovanovich, Bruce Leisz.
Application Number | 20220290092 15/734128 |
Document ID | / |
Family ID | 1000006560431 |
Filed Date | 2022-09-15 |
United States Patent
Application |
20220290092 |
Kind Code |
A9 |
Jovanovich; Stevan Bogdan ;
et al. |
September 15, 2022 |
METHOD AND APPARATUS FOR PROCESSING TISSUE SAMPLES
Abstract
A system, methods, and apparatus are described to collect and
prepare single cells, nuclei, subcellular components, and
biomolecules from specimens including tissues and in some
embodiments use the single cells to form organoids or microtissues.
The system can perform enzymatic and/or physical disruption of the
tissue to dissociate it into single-cells and then use a hanging
droplet method to form organoids or microtissues.
Inventors: |
Jovanovich; Stevan Bogdan;
(Livermore, CA) ; Chear; Kaiwan; (Livermore,
CA) ; Leisz; Bruce; (San Jose, CA) ; Eberhart;
David; (Santa Clara, CA) ; Bashkin; John;
(Fremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
S2 Genomics, Inc. |
Livermore |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20210214673 A1 |
July 15, 2021 |
|
|
Family ID: |
1000006560431 |
Appl. No.: |
15/734128 |
Filed: |
June 1, 2019 |
PCT Filed: |
June 1, 2019 |
PCT NO: |
PCT/US19/35097 |
371 Date: |
December 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62679494 |
Jun 1, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2001/4088 20130101;
G01N 1/31 20130101; C12M 45/06 20130101; C12M 45/20 20130101; G01N
1/286 20130101; G01N 1/4077 20130101; C12M 45/02 20130101; C12M
41/12 20130101; C12M 45/09 20130101 |
International
Class: |
C12M 1/33 20060101
C12M001/33; C12M 1/00 20060101 C12M001/00; C12M 1/34 20060101
C12M001/34; G01N 1/40 20060101 G01N001/40; G01N 1/31 20060101
G01N001/31; G01N 1/28 20060101 G01N001/28 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT (IF
ANY)
[0002] This research was supported in part by the National Human
Genome Research Institute of the National Institutes of Health
under award number RH010129.
Claims
1. A system comprising: (a) an instrument comprising: (i) one or
more cartridge interfaces, each configured to engage a cartridge
and comprising one or more fluid ports; (ii) a fluidics subsystem
comprising: (1) one or more sources of liquids and/or gasses; (2)
one or more fluid lines communicating with the sources and with
fluid ports in the cartridge interface; and (3) one or more pumps
configured to move liquids and/or gasses from the sources into
and/or out of the one or more fluid ports; (iii) a subsystem
comprising an actuator to actuate a tissue disruptor in a cartridge
engaged with a cartridge interface (e.g., a mechanical, pneumatic,
electromagnetic, or hydraulic actuator); and (b) one or more
cartridges, each engaged with one of the cartridge interfaces,
wherein each cartridge comprises: (i) one or more cartridge ports
communicating with the fluid ports in the cartridge interface; (ii)
a preprocessing chamber comprising an opening and, positioned in
the opening, a tissue disruptor configured for mechanical
disruption of tissue, wherein the tissue disruptor engages with and
is actuated by the actuator when the cartridge is engaged with the
cartridge interface; and (iii) a processing chamber communicating
with the preprocessing chamber, and with one or more cartridge
ports and configured to collect a suspension of biological material
from the preprocessing chamber.
2. The system of claim 1, wherein the instrument further comprises:
none, one or a plurality of valves configured to direct positive or
negative pressure from the one or more pumps through fluid lines
and/or the one or more containers connecting the one or more fluid
lines to the fluid ports.
3. The system of claim 1, wherein the instrument further comprises:
a magnetic processing module comprising a source of a magnetic
field, wherein the source is positioned to form a magnetic field in
a processing chamber of an engaged cartridge.
4. The system of claim 1, wherein the instrument further comprises:
a measurement subsystem.
5. The system of claim 1, wherein the instrument further comprises:
a control subsystem comprising a processor and memory, wherein the
memory comprises code that, when executed by the processor,
operates the system.
6. The system of claim 1, wherein the instrument further comprises:
a waste container communicating with the one or more pumps.
7. The system of claim 1, wherein the instrument further comprises:
a temperature subsystem configured to regulate temperature in a
chamber of the cartridge.
8. The system of claim 7, wherein the temperature subsystem
comprises a temperature regulating element (e.g., a Peltier, a
resistive heater, a circulating fluid), a controller to control the
temperature-regulating element and a thermal transfer element that
transfers heat from the temperature-regulating element to or from
the cartridge chambers.
9. The system of claim 1, wherein the actuator comprises a drive
head selected from slotted, phillips, quadrex, tri-wing, spanner
and hex.
10. The system of claim 1, wherein the at least one pump comprises
a syringe pump.
11. The system of claim 1, wherein the pump communicates through a
fluid line with a fluid port in the cartridge interface that
engages a cartridge port that communicates with the processing
chamber, wherein vacuum applied through the fluid line pulls fluid
from the preprocessing chamber into the processing chamber.
12. The system of claim 1, wherein the pump communicates through a
first fluid line with a container of fluid and with a second line
with a fluid port in the cartridge interface that engages a
cartridge port that communicates with the preprocessing chamber or
the processing chamber, wherein negative pressure applied through
the first fluid line pulls fluid from container and positive
pressure applied through the second fluid line pushes the fluid
into the preprocessing chamber or the processing chamber.
13. The system of claim 1, wherein each cartridge interface further
comprises a reagent inlet port that communicates with a port in the
preprocessing chamber configured to introduce reagent directly into
the prepossessing chamber.
14. The system of claim 1, wherein the preprocessing chamber
communicates with the processing chamber directly through a fluid
line, or indirectly, through one or more fluid lines in the
interface that communicate with ports in each of the preprocessing
chamber and the processing chamber.
15. The system of claim 1, wherein the preprocessing chamber
comprises no direct cartridge ports.
16. The system of claim 1, wherein the cartridge comprises no more
than any of one, two, three or four ports communicating with the
cartridge interface or with the environment.
17. The system of claim 1, wherein the cartridge comprises a
plurality of ports communicating with the cartridge interface or
with the environment, wherein at least one port is covered by a
cap.
18. The system of claim 1, wherein the tissue disruptor comprises:
(i) a sheath, (ii) a plunger configured to move slidably within the
sheath and comprising a coupler to engage the actuator and a head
for disrupting tissue, and (iii) a bias mechanism, e.g., spring,
that biases the plunger toward a retracted position, i.e. wherein
actuation is required to actuate the plunger to a forward
position.
19. The system of claim 18, wherein the plunger also can rotate
around the longitudinal axis of the sheath.
20. The system of claim 18, wherein the head has a circumference
which, when the head moves within the preprocessing chamber,
provides a gap between the head and a wall of the preprocessing
chamber between about 25 microns and 400 microns, e.g., sufficient
to allow cells, nuclei or microstructures of cells to pass between
the head and the wall without rupturing.
21. The system of claim 18, wherein the head comprises a disruption
surface comprising raised features for disrupting tissue.
22. The system of claim 18, wherein the plunger comprises a top
side comprising a feature for engaging the actuator.
23. The system of claim 1, wherein the tissue disrupter is seated
on a seat, e.g., an annular seat, in the preprocessing chamber.
24. The system of claim 18, wherein the tissue disrupter is seated
on a seat, e.g., an annular seat, in the preprocessing chamber.
25. The system of claim 24, wherein the tissue disrupter head
comprises an annular bevel, and the seat in the preprocessing
chamber is configured to mate with the bevel such that when the
plunger is actuated toward the bottom of the preprocessing chamber,
there is a defined annular gap between the head and a wall of the
preprocessing chamber, and no gap or a defined minimum gap between
the disruption surface of the head and the bottom of the
preprocessing chamber.
26. The system of claim 1, wherein the preprocessing chamber
comprises a bottom surface comprising raised features for
disrupting tissue.
27. The system of claim 1, wherein the preprocessing chamber
communicates with the processing chamber through a fluidic channel
connecting a port in a side of the preprocessing chamber with a
port in a top of the processing chamber.
28. The system of claim 1, wherein the processing chamber further
comprises a strainer (e.g., filter or a mesh screen) positioned to
strain suspension of biological material entering the processing
chamber from the preprocessing chamber.
29. The system of claim 1, wherein the processing chamber
communicates with a cartridge port configured such that when vacuum
is applied to the cartridge port, liquid in the preprocessing
chamber is pulled into the processing chamber.
30. The system of claim 29, wherein the cartridge further comprises
a vacuum trap fluidically connected with and positioned between the
cartridge port with the processing chamber.
31. The system of claim 1, wherein the processing chamber comprises
a drain section and a more narrow slot section and wherein the
processing chamber comprises a slanted floor configured to direct
fluid in the drain section toward the slot section.
32. The system of claim 1, wherein the processing chamber comprises
a depression for collecting a suspension of biological
material.
33. The system of claim 1, wherein the processing chamber
communicates with a cartridge port configured to introduce fluids
into the processing chamber.
34. The system of claim 1, wherein the processing chamber comprises
a cover comprising a port that communicates through a fluidic
channel with the preprocessing chamber.
35. In another embodiment, the preprocessing chamber comprises a
cover comprising a seal (e.g. a friable seal, or septum) that, when
removed or opened (e.g., punctured), allows access to the
preprocessing chamber.
36. The system of claim 1, wherein the processing chamber comprises
a cover comprising a seal (e.g. a friable seal) that, when removed
or opened (e.g., punctured), allows access to the processing
chamber.
37. The system of claim 1, wherein the cartridge further comprises:
one or more waste chambers fluidically connected with the
processing chamber.
38. The system of claim 1, wherein the cartridge further comprises
an identifier comprising information about the cartridge and/or its
use (e.g., a barcode, an RFID, an EE PROM), and wherein the
instrument comprises a reader for reading information in the
identifier.
39. The system of claim 1, wherein the one or more sources of
liquids and/or gasses are comprised in a fluidic subsystem.
40. A method comprising: (a) providing a system of claim 1, wherein
the prepossessing chamber comprises a tissue sample; (b) disrupting
the tissue sample by using the actuator to actuate the tissue
disrupter to produce a suspension of biological material; and (c)
using the fluidic subsystem to move the suspension of biological
material from the preprocessing chamber into the processing
chamber.
41. The method of claim 40, further comprising: (d) removing the
suspension of biological material from the processing chamber.
42. The method of claim 40, wherein the prepossessing chamber
further comprises one or more enzymes for digesting extracellular
matrix.
43. The method of claim 40, wherein the prepossessing chamber
further comprises one or more detergents for lysing cell
membranes.
44. The method of claim 40, wherein the prepossessing chamber
further comprises liquid having a viscosity that slows the rate of
degradation of RNA or other biomolecules during or after tissue
disruption.
45. The method of claim 40, wherein disrupting comprises
positioning a disruption surface of the head a defined distance
from a bottom surface of the preprocessing chamber and rotating the
head to disrupt tissue in the preprocessing chamber.
46. The method of claim 40, wherein disrupting comprises
positioning a disruption surface of the head with respect to a
bottom surface of the preprocessing chamber at a plurality of
different gap distances and, at each gap distance, rotating the
head.
47. The method of claim 46, wherein at at least one gap distance at
least some portion of the disruption head contacts some portion of
the bottom surface.
48. The method of claim 46, wherein the widest gap distance between
a flat portion of the surface and flat portion of the bottom of the
chamber is no more than any of 6 mm, 5 mm 4 mm, 3 mm, 2 mm, 1 mm,
500 .mu.m, 250 .mu.m, 100 .mu.m, 75 .mu.m, 50 .mu.m, 25 .mu.m, 20
.mu.m, 15 .mu.m, 10 .mu.m, 5 .mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1
um.
49. The method of claim 46, wherein the plurality of gap distances
between a flat portion of the grinding surface and flat portion of
the bottom of the chamber is any of 2, 3, 4, 5, 6, 7, 8, 9 or 10
and the largest gap distance is no more than any of 6 mm, 5 mm 4
mm, 3 mm, 2 mm, 1 mm, 500 .mu.m, 250 .mu.m, 100 .mu.m, 75 .mu.m, 50
.mu.m, 25 .mu.m, 20 .mu.m, 15 .mu.m, 10 .mu.m, 5 .mu.m, 4 .mu.m, 3
.mu.m, 2 .mu.m, or 1 um.
50. The method of claim 46, comprising: disrupting tissue with the
tissue disruptor; incubating the disrupted tissue with at least one
enzyme that digests extracellular matrix; and disrupting the
incubated tissue with the tissue disruptor.
51. The method of claim 40, wherein the fluidic subsystem applies a
vacuum to a cartridge port communicating with the processing
chamber to move the suspension of biological material.
52. The method of claim 40, wherein the cartridge further comprises
a strainer and the suspension of biological material entering the
processing chamber is strained to remove particulate matter.
53. The method of claim 40, further comprising, after moving the
suspension of biological material, using the fluidics subsystem to
introduce a liquid into the preprocessing chamber through a
cartridge port and then using the fluidics subsystem to move the
liquid into the processing chamber.
54. The method of claim 40, further comprising, using the fluidics
subsystem to introduce one or more liquids comprising one or more
reagents through a cartridge port into the processing chamber.
55. The method of claim 54, wherein the reagent comprises an enzyme
or a particle comprising a binding agent (e.g., a binding agent
directed against a target on a cell surface or a surface of a
nucleus, virus or other biological target).
56. The method of claim 40, wherein the tissue comprises a target
cell and the method further comprises: contacting the suspension of
biological material in the processing chamber with solid particles
comprising binding agents that bind to the target cells and
sequester bound target cells within the suspension of biological
material.
57. The method of claim 56, further comprising separating the bound
target cells from the suspension.
58. The method of claim 56, wherein the tissue is tumor tissue and
the target cells are tumor infiltrating lymphocytes.
59. The method of claim 56, wherein the target cells are stem cells
or partially differentiated cells.
60. The method of claim 40, further comprising: determining the
expression of one or more genes in cells, nuclei, mitochondria or
other organelles of the suspension of biological material.
61. The method of claim 60, wherein the one or more genes is a
panel comprising a plurality of genes.
62. The method of claim 61, wherein the panel comprises genes
distinguishing a target cell type, e.g., hepatocytes, neurons,
kidney glomerulus parietal cell, cardiomyocytes.
63. The method of claim 61, wherein the panel comprises genes
distinguishing a CRISPR modified target cell.
64. The method of claim 61, wherein the panel comprises genes that
are differentially expressed when cells experience stress, e.g.,
anoikis.
65. The method of claim 61, comprising preparing a suspension of
biological material on each of a plurality of tissue samples under
different tissue disruption conditions, and identifying conditions
that produce cells or nuclei having a gene expression profile
closest to or further away from that of cells in the pre-disrupted
tissue sample.
66. The method of claim 61, wherein the panel comprises one or more
housekeeping genes, e.g., Actb, gapdh.
67. A cartridge comprising: (i) a preprocessing chamber comprising:
(1) an opening and, positioned in the opening, a tissue disruptor
configured for mechanical disruption of tissue, and (2) a
preprocessing chamber port; and (ii) a processing chamber
comprising a processing chamber port communicating with the
preprocessing chamber port through a fluid line, and (iii) a
cartridge port that communicates with the processing chamber,
wherein a vacuum applied to the cartridge port pulls material from
the preprocessing chamber into the processing chamber.
68. The cartridge of claim 67, wherein the cartridge port
communicates with the processing chamber directly or through a
vacuum trap.
69. A cartridge comprising: (i) a preprocessing chamber comprising
an opening and, positioned in the opening, a tissue disruptor
configured for mechanical disruption of tissue; (ii) a strain
chamber comprising a strainer, wherein the strain chamber
communicates with the preprocessing chamber; (iii) a first
processing chamber communicating with the strain chamber; (iv) an
optional second processing chamber communicating with the first
processing chamber; (v) one or more cartridge ports communicating
with the processing chamber and the second processing chamber if
present.
70. The cartridge of claim 69, further comprising: (vi) one or more
waste chambers communicating with the first processing chamber and
second processing chamber when present.
71. The cartridge of claim 69, wherein the first processing chamber
comprises an element (e.g., a nozzle) configured to produce a
hanging drop of liquid from the strain chamber.
72. A method of creating a microtissue comprising: (a) providing a
cartridge of claim 69 comprising a tissue; (b) disrupting the
tissue with the tissue disruptor to produce a cell suspension; (c)
straining the cell suspension with the strainer to produce strained
cell suspension; and (d) forming a hanging drop from the strained
cell suspension using the element.
73. The method of claim 72, wherein the microtissue is an
organoid.
74. The method of claim 72, further comprising: after forming the
hanging drop, adding a liquid or gas to the processing chamber to
support survival of the cells in the hanging drop.
75. A system comprising: (a) an instrument comprising: (i) one or
more cartridge interfaces, each configured to engage a cartridge
and comprising one or more fluid ports; (ii) a module comprising an
actuator to actuate a tissue disruptor in a cartridge engaged with
a cartridge interface (e.g., a mechanical, pneumatic,
electromagnetic, or hydraulic actuator); and (b) one or more
cartridges, each engaged with one of the cartridge interfaces,
wherein each cartridge comprises: (i) a preprocessing chamber
comprising an opening and, positioned in the opening, a tissue
disruptor configured for mechanical disruption of tissue.
76. The system of claim 75, wherein the cartridge does not include
any chambers other than the preprocessing chamber.
77. A cartridge comprising: (i) a preprocessing chamber comprising
an opening and, positioned in the opening, a tissue disruptor
configured for mechanical disruption of tissue.
78. A tissue disruptor comprising: (i) a sheath, (ii) a plunger
configured to move slidably within the sheath and comprising a
coupler to engage the actuator and a head for disrupting tissue,
and (iii) a bias mechanism, e.g., spring, that biases the plunger
toward a retracted position, i.e., wherein actuation is required to
actuate the plunger to a forward position.
79. The tissue disruptor of claim 78, wherein the sheath comprises
a seater element adapted to seat the tissue disruptor on a
seat.
80. The tissue disruptor of claim 78, wherein the seater element
comprises a flange adapted to sit on an annular ring.
81. The tissue disruptor of claim 78, wherein the seater element
comprises one or more tabs adapted to sit in one or more slots.
82. A method of creating a microtissue comprising: (a) providing a
cartridge of as described herein comprising a tissue; (b)
disrupting the tissue with the tissue disruptor to produce a cell
suspension; (c) straining the cell suspension with the strainer to
produce strained cell suspension; (d) selecting stem cells from
strained cell suspension; and (e) removing or growing the selected
stem cells in the cartridge.
83. A method of creating a microtissue comprising: (a) providing a
cartridge of as described herein comprising a tissue; (b)
disrupting the tissue with the tissue disruptor to produce a cell
suspension; (c) straining the cell suspension with the strainer to
produce strained cell suspension; (d) differentiating cells from
strained cell suspension into stem cells; and (e) growing or
removing the differentiated stem cells in the cartridge.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the priority date of
U.S. provisional patent application 62/679,466, filed Jun. 1,
2018,
THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT IF THE
CLAIMED INVENTION WAS MADE AS A RESULT OF ACTIVITIES WITHIN THE
SCOPE OF A JOINT RESEARCH AGREEMENT
[0003] None.
REFERENCE TO A "SEQUENCE LISTING"
[0004] None.
BACKGROUND OF THE INVENTION
A) Field of Invention
[0005] This invention relates to the field of sample preparation
from biological materials. More specifically, the invention relates
to the processing of solid tissues into single cells, nuclei,
biomolecules, and processed samples for bioanalysis and the use of
single cells to form organoids and other microtissues.
B) Description of Related Art
[0006] Analysis of single cells and groups of cells is providing
information to dissect and understand how cells function
individually and unprecedented insight into the range of individual
cellular responses aggregated in ensemble measurements. Single cell
methods for electrophysiology, flow cytometry, imaging, mass
spectrometry (Lanni, E. J., et. al. J Am Soc Mass Spectrom. 2014;
25(11):1897-907.), microarray (Wang L and K A Janes. Nat Protoc.
2013; 8(2):282-301.), and Next Generation Sequencing (NGS) (Saliba
A. E., et. al. Nucleic Acids Res. 2014; 42(14):8845-60.) have been
developed and are driving an increased understanding of fundamental
cellular processes, functions, and interconnected networks. As the
individual processes and functions are understood and
differentiated from ensemble measurements, the individual
information can in turn lead to discovery of how network processes
among cells operate. The networks may be in tissues, organs,
multicellular organisms, symbionts, biofilms, surfaces,
environments, or anywhere cells live and interact.
[0007] Model systems of tissues are an important tool in the
understanding of tissue function, development, and regulation.
Improved model systems are needed to be developed for basic
research, companion diagnostics, and screening of compounds to
develop therapeutics. Historically, two dimensional culture of
model cell lines have been used to gain knowledge and model tissue
function, typically with only one cell type at a time. However,
solid tissues are three dimensional (3D) structures with complex
interactions between multiple cell types. The two dimensional model
can lack cell to cell interactions and the impacts of the
extracellular matrix on the cells.
[0008] 3D structures of tissues, which can be termed microtissues,
can be constructed using many techniques including hanging droplets
or using 3D supports comprised of Matricel or hydrogels to create
small spheroid and other shapes that can better mimic actual
tissues. Typically the size is limited by the diffusion of
nutrients into the 3D structure. In some cases, the 3D is created
incorporating stem cells and can be termed an organoid (N. de
Souza, Organoids. Nature Methods volume 15, page 23 (2018); "Method
of the Year 2017: Organoids" Nature Methods 2018/01/03/online,15
http://dx.doi.org/10. 1038/nmeth.4575. 10.1038/nmeth.4575; Yin,
Xiaolei et al. Engineering Stem Cell Organoids, Cell Stem Cell ,
Volume 18, Issue 1, 25-38). While organoids are a promising
technology, the creation of organoids suffers from process
variablity and the use of cells typically not derived directly from
tissue. This invention solves the problems of releasing cells from
solid tissues for multiple applications including growing cells on
2D surfaces, 3D organoids, and cell suspensions.
[0009] Next Generation Sequencing (NGS) of single cells is rapidly
changing the state of knowledge of cells and tissue, discovering
new cell types, and increasing understanding of the diversity of
how cells and tissue function. Single cell NGS RNA sequencing
(Saliba A. E., et. al., Nucleic Acids Res. 2014; 42(14):8845-60.)
(Shapiro E. et. al., Nat Rev Genet. 2013; 14(9):618-30.) is
unveiling the complexity of cellular expression, and the
heterogenity from cell to cell, and from cell type to cell type
(Buettner F. et. al., Nat Biotechnol. 2015; 33(2):155-60.). In situ
sequencing (Ke R et. al., Nat Methods. 2013; 10(9):857-60.), (Lee J
H, et. al., Nat Protoc. 2015; 10(3):442-58.) (Lee J H, et. al.,
Science. 2014, 21; 343(6177):1360-3.) has shown the feasability of
directly sequencing fixed cells. However, for RNA, many fewer reads
are generated with in situ sequencing, biasing against detection of
low abundant transcripts. Photoactivatable tags have been used to
capture mRNA from single cells (Lovatt, D., et. al., Nat Methods.
2014; 11(2):190-6.) from known locations in tissue, albeit with low
throughput capture and manual cell collection.
[0010] The NGS market has grown explosively over the last 10 years
with cost reductions and throughput increases exceeding Moore's
law. The applications have expanded from whole genome sequencing to
RNA-Seq, ChIP-Seq, exome sequencing, to now single-cell sequencing,
single nuclei sequencing, ATAC-Seq, and many other exciting
applications. The power and low cost of NGS is broadly changing
life sciences and moving into translational medicine and the clinic
as precision medicine begins. Until recent years essentially all of
the NGS analysis was of `bulk samples` where the nucleic acids of
numerous cells had been pooled. There is a need for systems that
integrate the sample preparation of single-cell suspensions, and
single-cell libraries, and bulk libraries starting from original
unprocessed fresh specimens as well as banks of frozen tissue and
Formalin-Fixed Paraffin-Embedded (FFPE) tissue. This instant
invention enables a system, cartridges, and processes to process
solid tissues from many types of specimens comprising single cells,
single nuclei, and nucleic acids.
[0011] Single-cell sequencing is rapidly changing the state of
knowledge of cells and tissue, discovering new cell types, and
increasing the understanding of the diversity of how cells and
tissue function. Single-cell RNA sequencing (Shapiro E. Biezuner T,
Linnarsson S. Single-cell sequencing-based technologies will
revolutionize whole-organism science. Nat Rev Genet. 2013;
14(9):618-30. PMID: 23897237) has highlighted the complexity of
cellular expression, and the large heterogeneity from cell-to-cell,
and from cell type-to-cell type (Buettner F. Natarajan K N, Casale
F P, Proserpio V, Scialdone A, Theis F J, Teichmann S A, Marioni J
C, Stegle O. Computational analysis of cell-to-cell heterogeneity
in single-cell RNA-sequencing data reveals hidden subpopulations of
cells. Nat Biotechnol. 2015; 33(2):155-60. PMID: 25599176).
Single-cell sequencing (Wang., Y. and N. E. Navin. Advanced and
Applications of single-cell sequencing technologies. Molecular
Cell. 2015. 58:598-609. PMID 26000845.) is being applied to
development, brain structure and function, tumor progression and
resistance, immunogenetics, and more.
[0012] Single cell nucleic acid sequencing technology and methods
using NGS and Next Next Generation Sequencing (NNGS), such as
nanopores, are rapidly evolving. Common components are
incorporation of a marker or barcode for each cell and molecule,
reverse transcriptase for RNA sequencing, amplification, and
pooling of sample for NGS and NNGS (collectively termed NGS)
library preparation and analysis. Starting with isolated single
cells in wells, barcodes for individual cells and molecules have
been incorporated by reverse transcriptase template switching
before pooling and polymerase chain reaction (PCR) amplification
(Islam S. et. al. Genome Res. 2011; 21(7):1160-7.) (Ramskold D. et.
al. Nat Biotechnol. 2012; 30(8):777-82.) or on a barcoded poly-T
primer with linear amplification (Hashimshony T. et. al. Cell Rep.
2012 Sep. 27; 2(3):666-73.) and unique molecular identifiers
(Jaitin D. A. et. al. Science. 2014; 343(6172):776-9.).
[0013] Pioneering work has used micronozzles (Geng T. et. al. Anal
Chem. 2014; 86(1):703-12) to produce nanodroplets to perform highly
parallel processing of mRNA from single cells with reverse
transcription incorporating cell and molecular barcodes from freed
primers (in Drop) (Klein A. M. et. al. Cell. 2015;
161(5):1187-201.) or primers attached to paramagnetic beads
(DropSeq) (Macosko E. Z. et. al. Cell. 2015; 161(5):1202-14.); the
lysis conditions and reverse transcriptase described by (Fekete R.
A. and A. Nguyen. U.S. Pat. No. 8,288,106. Oct. 16, 2012) are
incorporated by reference cited therein are incorporated by
reference, including instrumentation, chemistry, workflows,
reactions conditions, flowcell design, and other teachings. Both
inDrop and DropSeq are scalable approaches have change the scale
from 100s of cells previously analyzed to 1,000s and more.
[0014] Single-cell sequencing is now providing new information to
biologists, genomic scientists, and clinical practitioners, and the
single-cell market is growing explosively, perhaps the next great
disruption in life sciences and medicine. Multiple companies are
providing systems to take single-cell suspensions and create
Single-cell RNA sequencing (scRNA-Seq) libraries that are analyzed
by the robust NGS sequencing and analysis pipeline. No system
integrates the upstream process to produce single-cell suspensions
for NGS single-cell sequencing or has integrated from tissue to
single-cell or single nuclei libraries.
[0015] The production of single-cells or nuclei or nucleic acids
from solid and liquid tissue is usually performed manually with a
number of devices used without process integration. A combination
of gentle mechanical disruption with enzymatic dissociation has
been shown to produce single-cells with the highest viability and
least cellular stress response (Quatromoni J G, Singhal S,
Bhojnagarwala P, Hancock W W, Albelda S M, Eruslanov E. An
optimized disaggregation method for human lung tumors that
preserves the phenotype and function of the immune cells. J Leukoc
Biol. 2015 Jan.; 97(1):201-9. doi: 10.1189/jlb.5TA0814-373. Epub
2014 Oct. 30.).
[0016] Many manual protocols for dissociating different tissues
exist, for example, Jungblut M., Oeltze K., Zehnter I., Hasselmann
D., Bosio A. (2009). Standardized Preparation of Single-Cell
Suspensions from Mouse Lung Tissue using the gentleMACS
Dissociator. JoVE. 29, doi: 10.3791/1266; Stagg A J, Burke F, Hill
S, Knight S C. Isolation of Mouse Spleen Dendritic Cells.
Protocols, Methods in Molecular Medicine. 2001: 64: 9-22. Doi:
10.1385/1592591507.; Lancelin, W., Guerrero-Plata, A. Isolation of
Mouse Lung Dendritic Cells. J. Vis. Exp. (57), e3563, 2011. DOI :
10.3791/3563; Smedsrod B, Pertoft H. Preparation of pure
hepatocytes and reticuloendothelial cells in high yield from a
single rat liver by means of Percoll centrifugation and selective
adherence. J Leukocyte Biol. 1985: 38: 213-30.; Meyer J,
Gonelle-Gispert C, Morel P, Buhler L Methods for Isolation and
Purification of Murine Liver Sinusoidal Endothelial Cells: A
Systematic Review. PLoS ONE 11(3) 2016: e0151945.
doi:10.1371/journal.pone.0151945.; Kondo S. Scheef E A, Sheibani N,
Sorenson C M. "PECAM-1 isoform-specific regulation of kidney
endothelial cell migration and capillary morphogenesis", Am J
Physiol Cell Physiol 292: C2070-C2083, (2007); doi:
10.1152/ajpcell.00489.2006.; Ehler, E., Moore-Morris, T., Lange, S.
Isolation and Culture of Neonatal Mouse Cardiomyocytes. J. Vis.
Exp. (79), e50154, doi:10.3791/50154 (2013).; Volovitz I Shapira N,
Ezer H, Gafni A, Lustgarten M, Alter T, Ben-Horin I, Barzilai O,
Shahar T, Kanner A, Fried I, Veshchev I, Grossman R, Ram, Z. A
non-aggressive, highly efficient, enzymatic method for dissociation
of human brain-tumors and brain-tissues to viable single cells. BMC
Neuroscience (2016) 17:30 doi: 10.1186/512868-016-0262-y; F. E
Dwulet and M. E. Smith, "Enzyme composition for tissue
dissociation," U.S. Pat. No. 5,952,215, Sep. 14, 1999.
[0017] For example, solid tissue of interest is usually dissected
and then minced into 1-5 mm pieces by hand or a blender type of
disruptor is used. Enzymes or a mixture of enzymes, such as
collagenases, hydrauronadase, papain, proteases, DNase, etc., are
added and the specimen incubated, typically with shaking or
rotation to aid dissociation to prepare single cells or nuclei from
tissue. In many procedures, the specimen is triturated multiple
times or mechanically disrupted. The mechanical disruption may be
through orifices, grinding, homogenization, forcing tissue through
screens or filters, sonication, blending, bead-beating, rotors with
features that dissociate tissue, and other methods to physically
disrupt tissue to help produce single cells.
[0018] Following dissociation, in some embodiments the dissociated
sample is passed through a filter, such as a 70 .mu.m filter, to
retain clumps of cells or debris. The filtrate which contains
single cells or nuclei may be further processed to change the media
or buffer; add, remove, or deactivate enzymes; concentrate cells or
biomolecules, lyse red blood cells, or capture specific cell types.
The processing typically involves multiple steps of centrifugation
and resuspension, density gradients, or magnetic bead capture of
specific cell types using antibodies, or other affinity capture
ligands, or fluorescent cell-activated sorting (FACS), or other
methods. The titer and viability of the single-cell suspension is
usually determined using optical imaging with a microscope and
haemocytometer, or an automated instrument. In many cases, the
viability is determined using Trypan blue or fluorescent dyes.
Quality control can include characterization of the nucleic acids
by gel electrophoresis on an instrument such as a BioAnalyzer, or
the determination of the expression of certain genes using reverse
transcripatase and quantitative polymerase chain reaction
(RT-qPCR), or other relevant methods.
[0019] The rapid production of nuclei can give a snapshot of gene
expression (Habib N, Li Y, Heidenreich M, Swiech L, Avraham-Davidi
I, Trombetta J J, Hession C, Zhang F, Regev A. Div-Seq:
Single-nucleus RNA-Seq reveals dynamics of rare adult newborn
neurons. Science. 2016 Aug. 26; 353(6302):925-8. doi:
10.1126/science.aad7038. Epub 2016 Jul. 28.; Grindberg R V,
Yee-Greenbaum J L, McConnell M J, Novotny M, O'Shaughnessy A L,
Lambert G M, Ara zo-Bravo M J, Lee J, Fishman M, Robbins G E, Lin
X, Venepally P, Badger J H, Galbraith D W, Gage F H, Lasken R S.
RNA-sequencing from single nuclei. Proc Natl Acad Sci U S A. 2013
Dec. 3; 110(49):19802-7. doi: 10.1073/pnas.1319700110. Epub 2013
Nov. 18.).
[0020] The production of nuclei from tissue can be performed using
a Dounce homogenizer in the presence of a buffer with a detergent
that lyses cells but not nuclei. Nuclei can also be prepared
starting from single cell suspensions
(CG000124_SamplePrepDemonstratedProtocol_-_Nuclei_RevB, 10x
Genomics,
https://assets.contentful.com/an68im79xiti/6FhJX6yndYy0OwskGmMc8l/48c341c-
178fe
afa3ce21f5345ed3367b/CG000124_SamplePrepDemonstratedProtocol_-_Nucle-
i_RevB.pdf) by addition of a lysis buffer such as 10 mM Tris-HCl,
10 mM NaCl, 3 mM MgCl2 and 0.005% Nonidet P40 in nuclease-free
water and incubation for 5 min on ice before centifugation to
pellet the nuclei followed by resuspension in a resuspension buffer
such as 1X Phosphate Buffered Saline (PBS) with 1.0% BSA and 0.2
U/.mu.l RNase Inhibitor. The nuclei may be repeatedly pelleted and
resuspended to purify them or density gradients or other
purification methods used. The titer and viability of the nuclei
suspension is usually determined using optical imaging with a
microscope and haemocytometer, or an automated instrument with
viability determined using Trypan blue or fluorescent dyes.
[0021] The multi-process workflow to produce and characterize
single-cells and nuclei from tissue is a usually performed manually
using several devices without process integration, limiting the
scalablity of single cell sequencing and the integration with
downstream processes to create a sample-to-answer system. It is
laborious and requires skilled technicians or scientists, and
results in variability in the quality of the single-cells, and,
therefore, in the downstream libraries, analysis, and data. The
multiple steps and skill required can lead to differing qualities
of single cells or nuclei produced even from the same specimen,
limiting clinical utility. Today, the production of high quality
single-cells can take months of optimization.
[0022] Standarization is necessary before routine single-cell
preparation can be performed, particularly in clinical settings. In
addition, the length of the process and the process of dissociation
can lead to the tissue and cells changing physiology and altering
their expression of biomolecules such as RNA, proteins, lipids, and
metabolites in response to the stresses of the procedure,
accentuated by potentially long processing times. A crucial recent
insight is that cell processing methods, for example, the use of a
protease to dissociate cells from tissue (Lacar B, Linker S B,
Jaeger B N, Krishnaswami S, Barron J, Kelder M, Parylak S, Paquola
A, Venepally P, Novotny M, O'Connor C, Fitzpatrick C, Erwin J, Hsu
J Y, Husband D, McConnell M J, Lasken R, Gage F H. Nuclear RNA-seq
of single neurons reveals molecular signatures of activation. Nat
Commun. 2016 Apr. 19; 7:11022. doi: 10.1038/ncomms11022. PMID:
27090946.) can alter gene expression by placing cells under stress,
confounding analysis of the true transcriptome.
[0023] Robust, automated sample preparation is required to simplify
workflows before full process or physical integration with
downstream NGS analysis can be achieved to produce true
sample-to-answer solid tissue to single cell/nuclei NGS analysis
systems in the future. Robust processes and automated systems are
required that will input a wide range of tissues from a wide range
of organisms and tissues and produce high-quality single-cell or
nuclei suspensions without intervention, at acceptable viability
for suspensions, with minimal changes to gene expression
patterns.
[0024] To achieve a standardized process will require a system that
automates the sample preparation of cells or nuclei from tissue
with a single-use disposable cartridge. In some cases, microvalves
can be used in cartridges. Microvalves are comprised of mechanical
(thermopneumatic, pneumatic, and shape memory alloy),
non-mechanical (hydrogel, sol-gel, paraffin, and ice), and external
(modular built-in, pneumatic, and non-pneumatic) microvalves (as
described in: C. Zhang, D. Xing, and Y. Li., Micropumps,
microvalves, and micromixers within PCR microfluidic chips:
Advances and trends. Biotechnology Advances. Volume 25, Issue 5,
September-October 2007, Pages 483-514; Diaz-Gonzalez M., C.
Fernandez-Sanchez, and A. Baldi A. Multiple actuation microvalves
in wax microfluidics. Lab Chip. 2016 Oct. 5; 16(20):3969-3976.; Kim
J., Stockton A M, Jensen E C, Mathies R A. Pneumatically actuated
microvalve circuits for programmable automation of chemical and
biochemical analysis. Lab Chip. 2016 Mar. 7; 16(5):812-9. doi:
10.1039/c51c01397f; Samad M F, Kouzani A Z. Design and analysis of
a low actuation voltage electrowetting-on-dielectric microvalve for
drug delivery applications. Conf Proc IEEE Eng Med Biol Soc. 2014;
2014:4423-6. doi: 10.1109/EMBC.2014.6944605.; Samad M F, Kouzani A
Z. Design and analysis of a low actuation voltage
electrowetting-on-dielectric microvalve for drug delivery
applications. Conf Proc IEEE Eng Med Biol Soc. 2014; 2014:4423-6.
doi: 10.1109/EMBC.2014.6944605.; Lee E, Lee H, Yoo SI, Yoon J.
Photothermally triggered fast responding hydrogels incorporating a
hydrophobic moiety for light-controlled microvalves. ACS Appl Mater
Interfaces. 2014 Oct. 8; 6(19):16949-55. doi: 10.1021/am504502y.
Epub 2014 Sep. 25.; Liu X, Li S. An electromagnetic microvalve for
pneumatic control of microfluidic systems. J Lab Autom. 2014 Oct.;
19(5):444-53. doi: 10.1177/2211068214531760. Epub 2014 Apr. 17;
Desai A V, Tice J D, Apblett C A, Kenis P J. Design considerations
for electrostatic microvalves with applications in
poly(dimethylsiloxane)-based microfluidics. Lab Chip. 2012 Mar. 21;
12(6):1078-88. doi: 10.1039/c21c21133e. Epub 2012 Feb 3.; Kim J,
Kang M, Jensen E C, Mathies R A Lifting gate polydimethylsiloxane
microvalves and pumps for microfluidic control. Anal Chem. 2012
Feb. 21; 84(4):2067-71. doi: 10.1021/ac202934x. Epub 2012 Feb. 1;
Lai H, Folch A. Design and dynamic characterization of
"single-stroke" peristaltic PDMS micropumps. Lab Chip. 2011 Jan.
21; 11(2):336-42. doi: 10.1039/c01c00023j. Epub 2010 Oct. 19). The
system embodiments described herein can operate cartridges with no
valves, or valves can be incorporated into the cartridges to direct
flow.
[0025] Fluidic connections between cartridges and the instrument
fluidics can be achieved by the use of spring-loaded connectors and
modular microfluidic connectors as taught by Jovanovich, S. B. et.
al. Capillary valve, connector, and router. Feb. 20, 2001. U. S.
Pat. No. 6,190,616 and Jovanovich; S. B. et. al. Method of merging
chemical reactants in capillary tubes, Apr. 22, 2003, U.S. Pat. No.
6,551,839; and Jovanovich, S., I. Blaga, and R. McIntosh.
Integrated system with modular microfluidic components. U.S. Pat.
No. 7,244,961. Jul. 17, 2007. which are incorporated by reference
and their teachings which describe the modular microfluidic
connectors and details of modular microfluidic connectors,
including their use as multiway valves, routers, and other
functions including microfluidic circuits to perform flowthrough
reactions and flow cells with internally reflecting surfaces.
[0026] The surface chemistries of the paramagnetic beads and
conditions to bind cells or precipitate, wash, and elute nucleic
acids and other biomolecules onto surfaces is well understood,
(Boom, W. R. et. al. U.S. Pat. No. 5,234,809. Aug. 10, 1993.),
(Reeve, M. and P. Robinson. U.S. Pat. No. 5,665,554. Sep. 9,
1997.), (Hawkins, T. U.S. Pat. No. 5,898,071. Apr. 27, 1999.),
(McKernan, K. et. al. U.S. Pat. No., 6,534,262. Mar. 18, 2003.),
(Han, Z. U.S. Pat. No., 8,536,322. Sep. 17, 2013.), (Dressman et
al., "Transforming single DNA molecules into fluorescent magnetic
particles for detection and enumeration of genetic variation" Proc.
Natl. Acad. Sci. 100(15):8817-8822 (2003)), (Ghadessy et al.,
"Directed evolution of polymerase function by compartmentalized
self-replication", Proc. Natl. Acad. Sci. 98(8):4552-4557 (2000)),
(Tawfik and Griffiths, "Man-made cell-like compartments for
molecular evolution" Nat. Biotech. 16(7):652-656 (1998)), (Williams
et al., "Amplification of complex gene libraries by emulsion PCR"
Nat. Meth. 3(7):545-550 (2006)), and many chemistries are possible
and within the scope of the instant disclosure.
BRIEF SUMMARY OF THE INVENTION
[0027] Disclosed herein is a Sample Processing System that
processes original or processed samples for bioanalysis or for the
development of microtissues or organoids. The Sample Processing
System processes are comprised of enzymatic and mechanical
disruption mechanisms with integrated fluidic processes. This
invention enables, among other things, the implementation of a
Sample Processing System that inputs solid, liquid, or gaseous
samples including tissue or other biological samples, and processes
the samples for bioanalysis and other analyses.
[0028] In some embodiments, the sample or specimen is a tissue
specimen. The tissue can be from any source such as a human,
animal, or plant tissue. Examples of tissues include, without
limitation, a biopsy sample, a core biopsy sample, a fine needle
aspirate, cellular conglomerate, an organ fragment, whole blood,
bone marrow, a biofilm, or any other solid, semi-solid, gelatinous,
frozen or fixed three dimensional or two dimensional cellular
matrix of biological . In another embodiment the released nucleic
acid is bound to a membrane, chip surface, bead, surface, flow
cell, or particle. The term specimen is used to mean samples and
tissue specimens.
[0029] In one embodiment the Sample Processing System is used for
tissue processing. A Tissue Processing System embodiment can be
implemented as a flexible, extensible system that can process solid
or liquid tissue and other samples into single cells, nuclei,
organelles, and biomolecules with mechanical and enzymatic or
chemical processes to produce single cells in suspension, nuclei,
subcellular components, and biomolecules such as macromolecules
comprised of nucleic acids, comprised of DNA and RNA; proteins;
carbohydrates; lipids; biomolecules with multiple types of
macromolecules; metabolites; and other biological components,
including natural products for bioanalysis in suspension, in
solution, or attached to a surface. In some embodiments, the Tissue
Processing System performs affinity or other purifications to
enrich or deplete cell types, organelles such as nuclei,
mitochondria, ribosomes, or other organelles, or extracellular
fluids. In some embodiments the Tissue Processing System can
perform NGS library preparation. In some embodiments, the Tissue
Processing System processes tissue into single-cell libraries for
sequencing including Sanger, NGS, NNGS and other nucleic acid
sequencing technolgies, protein sequencing, or protoeomics, or
other analytical methods.
[0030] Disclosed herein are different embodiments of Sample
Processing Systems that integrate two or more of the overall steps
to take samples from specimens (i.e., tissue, biofilms, other
multi-dimensional matrices with cells or viruses, liquids) and
prepare single cell or nuclei in suspensions or on surfaces, or
further process the specimens into biomolecules including
macromolecules comprised of nucleic acids, comprised of DNA and
RNA; proteins; carbohydrates; lipids; biomolecules with multiple
types of macromolecules; metabolites; and other biological
components, including natural products). In some embodiments
specimen can be processed into NGS sequencing libraries, or fully
integrated with an analytical system to produce a sample-to-answer
systems such as a sample-to-answer genomic system.
[0031] In some embodiments the Sample Processing System can be
integrated with downstream bioanalysis to create a sample-to-answer
system. In a preferred embodiment of the Sample Processing System,
a Tissue Processing System processing embodiment is integrated with
a nucleic acid bioanalysis system to sequence nucleic acids from
tissues. Integrated is used to mean the workflows directly
interface or in other contexts that the physical system directly
interfaces or is incorporated into a system, instrument, or device.
In one embodiment, the Tissue Processing System is integrated with
a nucleic acid sequencer to produce a sample-to-answer system.
[0032] In one embodiment the Tissue Processing System can be used
to create microtissues or organoids directly in a cartridge using
the hanging droplet method or other methods, or the output of the
system can be used as the starting point for creating microtissues
or organoids off of the cartridge.
[0033] The Sample Processing System can have multiple subsystems
and modules that perform processing or analysis. In a preferred
embodiment of the Sample Processing System, one or more cartridges
performs one or more steps in the processing workflow. In some
embodiments the cartridges have multiple processing sites such as
processing chambers that can process more than one sample. In some
embodiments a cap couples mechanical disruption on the cartridge
from a Physical Dissociation Subsystem to the Enzymatic and
Chemical Dissociation Subsystem in a processing chamber. In some
embodiments reagents from an Enzymatic and Chemical Dissociation
Subsystem are delivered to the cartridge by a Fluidic Subsystem to
regions that are used as Pre-Processing Chambers and Processing
Chambers to disrupt or dissociate the specimen and process the
cells, subcellular components, and biomolecules for
bioanalysis.
[0034] The addition of fluids can be controlled by a Fluidic
Subsystem with the complete system controlled by software in a
Control Subsystem which can include the user interface through a
device comprised of monitor, embedded display, touch screen; or
through audio commands through the system or an accessory devices
such as a cell phone or microphone. In some instances the Control
Subsytem can include interfaces to laboratory information
management systems, other instruments, databases, analysis
software, email, and other applications.
[0035] In some embodiments, the amount of dissociation is monitored
at intervals during the dissociation and in some instances the
viability determined during processing using a Measurement
Subsystem. The degree of dissociation and/or viability can be
determined inside the main dissociation compartment and/or in a
separate compartment or channel, and/or in the external
instrument.
[0036] In some embodiments, cell imaging solutions, such as cell
type specific antibodies, stains, or other reagents, can be added
to the tissue or single cells or nuclei for additional processing
or imaging. The imaging can capture cells, subcellular structures,
or histological or other data. In some embodiments the images can
be analyzed to direct the operation and workflow of the Sample
Processing System through decisions trees, hash tables, machine
learning, or artificial intelligence.
[0037] In some embodiments, single cells or nuclei in suspension or
on surfaces are further processed using magnetic bead or particle
technologies using a Magnetic Processing module to purify or
deplete cell types, nuclei, nucleic acids, or other
biomolecules.
[0038] The term singulated cells is used to mean single cells in
suspension or on a surface or in a well including a microwell or
nanowell such that they can be processed as single cells. The term
singulated cells is also used at times to encompass single
nuclei.
[0039] In one embodiment, the specimen is added to a cartridge
which performs both physical and enzymatic dissociation of the
tissue. In some embodiments the Singulator System performs
trituration and other physical dissociation modalities as a step or
steps in the process of singulating cells. The physical
dissociation modalities include passing the specimen through
screens, filters, orifices, grinding, blending, sonication,
smearing,bead beating, and other methods known to one skilled in
the art to physically disrupt tissue to help produce single cells
or nuclei or nucleic acids or other biomolecules.
[0040] In one embodiment, the specimen is added to a cartridge
which performs both physical and chemical dissociation of the
tissue into nuclei. In some embodiments the Singulator System
performs trituration and other physical dissociation modalities as
a step or steps in the process of producing nuclei suspensions. The
physical dissociation modalities include passing the specimen
through screens, filters, orifices, gaps, grinding, blending,
sonication, smearing,bead beating, and other methods known to one
skilled in the art to physically disrupt tissue to help produce
nuclei or nucleic acids or other biomolecules when using chemical
treatment of tissues.
[0041] In one embodiment, the Sample Processing System is a
Singulator System embodiment. The Singulator System described can
input raw, unprocessed samples, or other primary or secondary
samples, and output single cells or nuclei ready for single cell or
nuclei analysis or for additional processing, e.g., to purify
specific cell types with antibodies or by cell sorting or growth,
library preparation, or many other applications. A Singulator
System embodiment dissociates single cells or nuclei from specimens
such as tissue, blood, bodily fluid or other liquids or solids
containining cells to produce single cells in suspensions or
nuclei, or on surfaces, in matrices, or other output
configurations. In a preferred Singulation System described
embodiment, there is a cartridge that inputs tissue and/or other
specimens and outputs single cells or nuclei, preferably of known
titer in a buffer supplemented with media such as Hank's buffer
with 2% fetal calf serum.
[0042] In some embodiments, the Sample Processing System, such as a
Singulator System embodiment, uses enzymes to assist in the process
of singulating cells including enzymes to preserve nucleic acids
and prevent clumping. The enzymes and additives are comprised of
but not limited to collagenases (e.g., collagenases type I, II,
III, IV, and others), elastase, trypsin, papain, tyrpLE,
hyaluronidase, chymotrypsin, neutral protease, pronase, liberase,
clostripain, caseinase, neutral protease (Dispase.RTM.), DNAse,
protease XIV, RNase inhibitors, or other enzymes, protease
inhibitors, active site inhibitors, EDTA, EGTA, biochemicals, or
chemicals such as Triton X-100, Nonidet P40, detergents,
surfactants, etc. In other embodiments, different reagents or
mixtures of reagents are applied sequentially to dissociate the
biological sample or specimen into single-cell suspensions.
[0043] In some embodiments, the Sample Processing System, such as a
Singulator System embodiment, uses chemicals, enzymes, or both to
assist in the process of producing nuclei from solid tissue in a
nuclei isolation solution, assist in tissue dissociation, to
preserve nucleic acids, and to prevent clumping. The chemicals are
comprised of but not limited to detergents, surfactants, non-ionic
surfactants, Triton X-100, Tween, Brij, CHAPS, Nonidet P40, Igepal,
glycosides, HEGA, MEGA, or digitonin; the enzymes are comprised of
collagenases (e.g., collagenases type I, II, Ill, IV, and others),
elastase, trypsin, papain, tyrpLE, hyaluronidase, chymotrypsin,
neutral protease, pronase, liberase, clostripain, caseinase,
neutral protease (Dispase.RTM.), DNAse, protease XIV, or other
enzymes. In some embodiments inhibitors such as RNase inhibitors,
protease inhibitors, active site inhibitors, or biochemicals that
sequester or chelate ions essential for RNases, comprising EDTA or
EGTA or sodium citrate, can be used in solutions. In some
embodiments, spermine or spermidine or sodium butyrate, or sodium
orthovanadate, or sodium fluoride are included in the nuclei
isolation solution or in nuclei storage solutions. In other
embodiments, different reagents or mixtures of reagents are applied
sequentially to dissociate the biological sample or specimen into
single-cell suspensions and then the single cells are processed
into nuclei. In some embodiment, the viscosity of the solutions are
increased using chemicals comprised of ficoll, or gylcerol, or
dextran, or sucrose, or trehalose, or polyethylene glycol, or
cellulose or other compounds to slow diffusion rates of RNases or
DNases or other enzymes or compounds that degrade biomolecules. In
some embodiments the counterions in the buffers are acetate.
[0044] In some embodiments the Singulator System produces cell
suspensions of known titers and viability. In some embodiments the
Singulator System produces nuclei suspensions of known titers and
quality. In some embodiments the Singulator System monitors the
viability and/or the amount of singulation of a sample and adjusts
the treatment time and concentration of enzymes or other
dissociation agents by monitoring of the dissociation, for example
by the production of single cells or nuclei. The monitoring can be
in real time, in intervals, or endpoints or any combinations
thereof.
[0045] The Singulator System can in some embodiments select from
sets of reagents to dissociate tissue and adjust according to
production of single cells or viability of cells as monitored by
the system, in some instances in real time, at intervals, or as an
endpoint. The single-cell suspensions produced by the Singulator
System can be used to generate cells with therapeutic application,
e.g., re-grow new tissues and/or organs and/or organisms.
[0046] The Singulator System has advantages over existing
technology and can produce single cells, nuclei, or biomolecules
from tissue in an automated and standardized instrument that can in
some embodiments process the specimens into NGS libraries or other
preparations. The Singulator System will enable users, e.g.,
researchers, clinicians, forensic scientists, and many disciplines
to perform identical processing on biosamples, reducing user
variability, and throughput constraints of manual processing.
[0047] Embodiments of the Singulation System can prepare
single-cells or nuclei or nucleic acids for analysis by methods
comprised of DNA sequencing, DNA microarrays, RNA sequencing, mass
spectrometry, Raman spectroscopy, electrophysiology, flow
cytometry, mass cytometry, and many other analytical methods well
known to one skilled in the art including multidimensional analysis
(e.g., LC/MS, CE/MS, etc.). In addition, single-cell suspensions or
on surfaces or matrices can be used to grow additional cells
including genetically altered by methods such as CRISPR, engineered
viral or nucleic acid sequences, in tissue culture, or to grow
tissues or organs for research and therapeutic purposes.
[0048] The Singulator System embodiment described is compatible
with commercially available downstream library preparation and
analysis by both NGS and NNGS sequencers. The term NGS is used to
connote either NGS or NNGS sequencers or sample preparation methods
as appropriate. As contemplated herein, next generation sequencing
or next-next generation sequencing refers to high-throughput
sequencing, such as massivley parallel sequencing, (e.g.,
simultaneously (or in rapid succession) sequencing any of at least
1,000, 100,000, 1 million, 10 million, 100 million, or 1 billion
polynucleotide molecules). Sequencing methods may include, but are
not limited to: high-throughput sequencing, pyrosequencing,
sequencing-by-synthesis, single-molecule sequencing, nanopore
sequencing, semiconductor sequencing, sequencing-by-ligation,
sequencing-by-hybridization, RNA-Seq (Illumine), Digital Gene
Expression (Helicos), next generation sequencing, Single Molecule
Sequencing by Synthesis (SMSS) (Helicos), massively-parallel
sequencing, Clonal Single Molecule Array (Solexa), shotgun
sequencing, Maxam-Gilbert or Sanger sequencing, primer walking,
sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys), DNA
nanoball sequencing (Complete Genomics), or nanopore (e.g., Oxford
Nanopore, Roche) platforms and any other sequencing methods known
in the art.
[0049] In another aspect provided herein is an apparatus,
composition of matter, or article of manufacture, and any
improvements, enhancements, and modifications thereto, as described
in part or in full herein and as shown in any applicable Figures,
including one or more features in one or more embodiment.
[0050] In another aspect provided herein is an apparatus,
composition of matter, or article of manufacture, and any
improvements, enhancements, and modifications thereto, as described
in part of in full herein and as shown in any applicable Figures,
including each and every feature.
[0051] In another aspect provided herein is a method or process of
operation or production, and any improvements, enhancements, and
modifications thereto, as described in part or in full herein and
as shown in any applicable Figures, including one or more feature
in one or more embodiment.
[0052] In another aspect provided herein is a method or process of
operation or production, and any improvements, enhancements, and
modifications thereto, as described in part or in full herein and
as shown in any applicable Figures, including each and every
feature.
[0053] In another aspect provided herein is a product, composition
of matter, or article of manufacture, and any improvements,
enhancements, and modifications thereto, produced or resulting from
any processes described in full or in part herein and as shown in
any applicable Figures.
[0054] In one embodiment the single-cell suspension is prepared for
a bioanalysis module for downstream analysis including but not
limited to sequencing, next generation sequencing, next next
generation sequencing, protein sequencing, proteomic analysis,
genomic analysis, gene expression, gene mapping, carbohydrate
characterization and profiling, lipid characterization and
profiling, flow cytometry, imaging, DNA or RNA microarray analysis,
metabolic profiling, functional, or mass spectrometry, or
combinations thereof.
[0055] In another aspect provided herein is a data analysis system
that correlates, analyzes, stores, and visualizes the analytical
information of a sample component such as its viability, degree of
single cell or nuclei dissociation, with the processing step and
measures the change over time, and/or amount of enyzmatic activity,
and/or physical disruptions of the original biological specimen. In
another aspect provided herein is a data analysis system that
correlates, analyzes, stores, and visualizes the analytical
information of a sample component and shares metadata of the sample
with downstream or upstream laboratory information systems.
[0056] In another aspect provided herein is a data analysis system
that correlates, analyzes, and visualizes the analytical
information of a sample component such as its viability, degree of
single cell or nuclei dissociation, with the processing step and
measures the change over time, and/or amount of enyzmatic activity,
and/or physical disruptions of the original biological specimen and
adjusts the processing parameters from the analytical
information.
[0057] The Singulator System is a novel platform that automates and
standardizes the only portion of the single-cell NGS workflow that
has not been automated. This will have broad impacts. Process
standardization will be critical for comparison of data from lab to
lab or research to researcher. The Human Cell Atlas project intends
to freely share the multi-national results in an open database.
However, with no standardization of the complete process, direct
comparisons will greatly suffer from widely varying impacts of the
first processing step of producing single-cells or nuclei from
tissue. Additionally, when single-cell or nuclei sequencing becomes
clinically relevant, the standardization and de-skilling of the
production of single-cells or nuclei will be required to be
performed by an automated instrument such as the Singulator
System.
[0058] In another aspect, provided herein is a system comprising:
(a) an instrument comprising: (i) one or more cartridge interfaces
configured to engage a cartridge; (ii) a fluidic subsystem
comprising: (1) one or more containers containing one or more
liquids and/or gasses; (2) one or more fluid lines connecting the
containers with fluid ports in the cartridge interface; and (3) one
or more pumps configured to move liquids and/or gasses into and/or
out of the fluid port(s); (iii) one or more mechanical subsystems
comprising an actuator; (iv) optionally, one or more magnetic
processing modules comprising a source of magnetic force, wherein
the magnetic force is positioned to form a magnetic field in one or
more processing chambers; (v) optionally, one or more measurement
modules; (vi) optionally, one or more control subsystems comprising
a processor and memory, wherein the memory comprises code that,
when executed by the processor, operates the system; and (b) one or
more cartridges, each engaged with one of the cartridge interfaces,
wherein each cartridge comprises: (i) a sample inlet port; (ii) one
or more cartridge ports communicating with the fluid ports in the
cartridge interface; (iii) a preprocessing chamber communicating
with the sample inlet port and with at least one cartridge port,
and comprising a tissue disruptor configured for mechanical
disruption of tissue, wherein the tissue disruptor engages with and
is actuated by the actuator when the cartridge is engaged with the
cartridge interface; (iv) a strain chamber communicating with the
preprocessing chamber configured to separate cells and/or nuclei
from disrupted tissue optionally combined with either the
preprocessing or processing chambers; (v) a processing chamber
communicating with the strain chamber, optionally communicating
with one or more cartridge ports and configured to perform one or
more processing steps on separated cells and/or nuclei; and (vi)
optionally, one or more waste chambers fluidically connected with
the processing chamber. In one embodiment the tissue disruptor
comprises a grinder, a pestle or a variable orifice. In another
embodiment the system further comprises a barcode reader. In
another embodiment the system comprises a measurement module that
performs optical imaging to measure titer, clumping, and/or
viability of cells or nuclei or properties of biomolecules. In
another embodiment the system comprises a measurement module and a
control system, wherein the measurement module measures, and one or
more time points, characteristics of a sample in the processing
chamber, and control system comprises code that determines a state
of the sample, e.g., viability or degree of single cell or nuclei
dissociation, and that adjusts processing parameters. In another
embodiment the system further comprises (c) one or more analysis
modules, wherein an input port of the analysis module is in fluid
communication with the processing chamber. In another embodiment
the analysis module performs an analysis selected from one or more
of: DNA or RNA sequencing, next generation DNA or RNA sequencing,
next next generation DNA or RNA sequencing of nucleic acids and
their adducts such as epigenetic modifications; nanopore sequencing
of nucleic acids and their adducts, single cell DNA sequencing of
nucleic acids and their adducts; single nuclei RNA sequencing of
nucleic acids and their adducts; PCR, digital droplet PCR, qPCR,
RT-qPCR; genomic analysis, gene expression analysis, gene mapping,
DNA fragment mapping; imaging including optical and mass
spectrometry imaging; DNA or RNA microarray analysis; fluorescent,
Raman, optical, mass spectrometery and other detection modalities
of nucleic acids acids and their adducts with and without labels;
proteomic analysis, including fluorescent, Raman, optical, mass
spectrometery, protein sequencing, and other detection modalities
of proteins and peptides and their adducts and modifications;
carbohydrate characterization and profiling including sequencing,
fluorescent, Raman, optical, mass spectrometery, and other
detection modalities of carbohydrates and their adducts and other
covalent polymers; lipid characterization and profiling including
sequencing, fluorescent, Raman, optical, mass spectrometery, and
other detection modalities of lipids and their adducts and other
covalent polymers; flow cytometry; characterization of cells and
profiling including fluorescent, Raman, optical, mass
spectrometery, and other detection modalities of cells and their
adducts and other covalent polymers; metabolic profiling including
sequencing, fluorescent, Raman, optical, mass spectrometery, and
other detection modalities of metabolites and their adducts and
other covalent polymers; functional analysis including protein
protein interactions; bioinformatic analysis of cells, organelles,
and biomolecules; and mass spectrometry and other analytical
methods.
[0059] In another embodiment the cartridge interface comprises a
means of positioning the cartridge in the instrument that engages
the fluidic subsystem and the mechanical module and optionally is
temperature controlled. In another embodiment the cartridge is
disposable.
[0060] In another aspect provided herein is a method comprising:
(a) providing a tissue sample to a preprocessing chamber; (b)
automatically performing mechanical and enzymatic/chemical
disruption of the tissue in the preprocessing chamber to produce
disrupted tissue comprising released cells and/or nuclei and
debris; (c) automatically moving the disrupted tissue into a strain
chamber comprising a strainer and/or filter and separating the
released cells and/or nuclei from the debris therein; and (d)
automatically moving the released cells and/or nuclei into a
processing chamber which can be combined with the strain chamber in
a preferred embodiment. In another embodiment automatically moving
further comprises performing at least one processing step on the
released cells and/or nuclei in the processing chamber. In another
embodiment processing comprises one or more automatically performed
processes selected from:(I) lysing cells; (II) capturing cells;
(Ill) isolating nucleic acid; (IV) isolating protein; (V)
converting RNA into cDNA; (VI) preparing one or more libraries of
adapter tagged nucleic acids; (VII) performing PCR or other DNA
amplification methods; (VIII) isolating individual cells or
individual nuclei in nanodrops or nanoboluses or nanowells or in
media such as agarose that will limit diffusion of molecules of
interest between cells or nuclei; and (IX) outputting released
cells and/or nuclei into output vessels such as 8 well strip tubes,
microtiter plates, Eppendorf tubes, a chamber in the cartridge, or
other vessels capable of receiving cell suspensions. In another
embodiment the method further comprises: automatically capturing
the released cells and/or nuclei in the processing chamber by
binding to magnetically attractable particles comprising moieties
having affinity for the cells and/or nuclei and applying a magnetic
force to the processing chamber to immobilize the captured cells
and/or nuclei. In another embodiment the method further comprises:
automatically monitoring cell and/or nuclei titer in the
preprocessing chamber and, when the titer reaches a desired level,
exchanging a dissociation solution used to dissociate the tissue
for a buffer. In another embodiment the method further comprises
automatically monitoring a bioassay in the preprocessing or
processing chambers.
[0061] In another aspect provided herein is a cartridge comprising:
(i) a sample inlet port; (ii) optionally one or more cartridge
ports configured to communicate with fluid ports in a cartridge
interface; (iii) a preprocessing chamber communicating with the
sample inlet port and with at least one cartridge port, and
comprising a tissue disruptor configured for mechanical disruption
of tissue, wherein the tissue disruptor engages with and is
actuated by the actuator when the cartridge is engaged with the
cartridge interface; (iv) a strain chamber communicating with the
preprocessing chamber configured to separate cells from disrupted
tissue that can optionally be combined with the preprocessing or
processing chambers; (v) a processing chamber communicating with
the strain chamber, optionally communicating with one or more
cartridge ports and configured to perform one or more processing
steps on separated cells; and (vi) optionally, one or more waste
chambers fluidically connected with the processing chamber. In
another embodiment the cartridge further comprises a cap that opens
and closes the sample inlet port. In another embodiment the cap
comprises a tissue disruptor element that moves, for example, about
rotationally and back and forth along an axis. In another
embodiment the cartridge further comprises a holder. In another
embodiment the cartridge further comprises a top piece and a bottom
piece connected by collapsible element which allow the top piece
and/or the bottom piece to move relative to the holder. In another
embodiment the holder comprises a mesh screen. In another
embodiment the cartridge further comprises a grinding element for
grinding tissue in the preprocessing chamber. In another embodiment
the cartridge further comprises an identifier, such as a barcode or
other identification system that comprises information about the
cartridge and/or its use. In another embodiment the cartridge
further comprises a plunger configured to move slideably within the
preprocessing chamber. In another embodiment, the cartridge has one
or more valves.
[0062] In another aspect provided herein is a variable orifice
device for disrupting tissue comprising: (a) a first container and
a second container fluidically connected through a flexible tube
comprising a lumen; (b) an adjustable clamp positioned to clamp the
flexible tube, wherein adjusting the clamp alters the
cross-sectional area of the lumen; and (c) one or more pumps or
devices operatively coupled with the first and/or second containers
configured to push liquid in one container through the flexible
tubing into the other container. In another embodiment the
adjustable clamp comprises an eccentric cam operatively coupled to
a motor, wherein rotating the cam closes or opens the clamp.
[0063] In another aspect provided herein is a method for disrupting
tissue comprising: (a) providing a variable orifice device
comprising first container and a second container fluidically
connected through a flexible tube comprising a lumen; (b) moving a
sample comprising tissue from one of the containers through the
flexible tube to another one of the containers; (c) decreasing the
cross-sectional area of the lumen and moving the sample from one of
the containers through the flexible tube to another one of the
containers; (d) repeating step (c) one or more times to disrupt the
tissue.
[0064] In another aspect provided herein is a system comprising:
(a) an instrument comprising: (i) one or more cartridge interfaces,
each configured to engage a cartridge and comprising one or more
fluid ports; (ii) a fluidics subsystem comprising: (1) one or more
sources of liquids and/or gasses; (2) one or more fluid lines
communicating with the sources and with fluid ports in the
cartridge interface; and (3) one or more pumps configured to move
liquids and/or gasses from the sources into and/or out of the one
or more fluid ports; (iii) a subsystem comprising an actuator to
actuate a tissue disruptor in a cartridge engaged with a cartridge
interface (e.g., a mechanical, pneumatic, electromagnetic, or
hydraulic actuator); and (b) one or more cartridges, each engaged
with one of the cartridge interfaces, wherein each cartridge
comprises: (i) one or more cartridge ports communicating with the
fluid ports in the cartridge interface; (ii) a preprocessing
chamber comprising an opening and, positioned in the opening, a
tissue disruptor configured for mechanical disruption of tissue,
wherein the tissue disruptor engages with and is actuated by the
actuator when the cartridge is engaged with the cartridge
interface; and (iii) a processing chamber communicating with the
preprocessing chamber, and with one or more cartridge ports and
configured to collect a suspension of biological material from the
preprocessing chamber. In one embodiment, the instrument further
comprises: none, one or a plurality of valves configured to direct
positive or negative pressure from the one or more pumps through
fluid lines and/or the one or more containers connecting the one or
more fluid lines to the fluid ports. In another embodiment, the
instrument further comprises: a magnetic processing module
comprising a source of a magnetic field, wherein the source is
positioned to form a magnetic field in a processing chamber of an
engaged cartridge. In another embodiment, the instrument further
comprises: a measurement subsystem. In another embodiment, the
instrument further comprises: a control subsystem comprising a
processor, memory, and a local or remote database wherein the
memory comprises code that, when executed by the processor,
operates the system and can store information of the instrument
processes or analytical results from the system in a database. In
another embodiment, the instrument further comprises: a waste
container communicating with the one or more pumps. In another
embodiment, the instrument further comprises: a temperature
subsystem configured to regulate temperature in a chamber of the
cartridge. In another embodiment, the temperature subsystem
comprises a temperature regulating element (e.g., a Peltier, a
resistive heater, a circulating fluid), a controller to control the
temperature-regulating element and a thermal transfer element that
transfers heat from the temperature-regulating element to or from
the cartridge chambers. In another embodiment, the temperature
subsystem comprises a temperature regulating element (e.g., a
Peltier, a resistive heater, a circulating fluid), a controller to
control the temperature-regulating element and a thermal transfer
element that transfers heat from the temperature-regulating element
to or from the reagents and reagent containers. In another
embodiment, the actuator comprises a drive head selected from
slotted, phillips, quadrex, tri-wing, spanner and hex. In another
embodiment, the at least one pump comprises a syringe pump. In
another embodiment, the pump communicates through a fluid line with
a fluid port in the cartridge interface that engages a cartridge
port that communicates with the processing chamber, wherein vacuum
applied through the fluid line pulls fluid from the preprocessing
chamber into the processing chamber. In another embodiment, the
pump communicates through a first fluid line with a container of
fluid and with a second line with a fluid port in the cartridge
interface that engages a cartridge port that communicates with the
preprocessing chamber or the processing chamber, wherein negative
pressure applied through the first fluid line pulls fluid from
container and positive pressure applied through the second fluid
line pushes the fluid into the preprocessing chamber or the
processing chamber. In another embodiment, each cartridge interface
further comprises a reagent inlet port that communicates with a
port in the preprocessing chamber configured to introduce reagent
directly into the prepossessing chamber. In another embodiment, the
preprocessing chamber communicates with the processing chamber
directly through a fluid line, or indirectly, through one or more
fluid lines in the interface that communicate with ports in each of
the preprocessing chamber and the processing chamber. In another
embodiment, the preprocessing chamber comprises no direct cartridge
ports. In another embodiment, the cartridge comprises no more than
any of one, two, three or four ports communicating with the
cartridge interface or with the environment. In another embodiment,
the cartridge comprises a plurality of ports communicating with the
cartridge interface or with the environment, wherein at least one
port is covered by a cap. In another embodiment, the tissue
disruptor comprises: (i) a sheath, (ii) a plunger configured to
move slidably within the sheath and comprising a coupler to engage
the actuator and a head for disrupting tissue, and (iii) a bias
mechanism, e.g., spring, that biases the plunger toward a retracted
position, i.e. wherein actuation is required to actuate the plunger
to a forward position. In another embodiment, the plunger also can
rotate around the longitudinal axis of the sheath. In another
embodiment, the head has a circumference which, when the head moves
within the preprocessing chamber, provides a gap between the head
and a wall of the preprocessing chamber between about 25 microns
and 400 microns, e.g., sufficient to allow cells or nuclei or
microstructures of cells to pass between the head and the wall
without rupturing. In another embodiment, the head comprises a
disruption surface comprising raised features for disrupting
tissue. In another embodiment, the plunger comprises a top side
comprising a feature for engaging the actuator. In another
embodiment, the tissue disrupter is seated on a seat, e.g., an
annular seat, in the preprocessing chamber. In another embodiment,
the tissue disrupter head comprises an annular bevel, and the seat
in the preprocessing chamber is configured to mate with the bevel
such that when the plunger is actuated toward the bottom of the
preprocessing chamber, there is a defined annular gap between the
head and a wall of the preprocessing chamber, and no gap or a
defined minimum gap between the disruption surface of the head and
the bottom of the preprocessing chamber. In another embodiment, the
preprocessing chamber comprises a bottom surface comprising raised
features for disrupting tissue. In another embodiment, the
preprocessing chamber communicates with the processing chamber
through a fluidic channel connecting a port in a side of the
preprocessing chamber with a port in a top of the processing
chamber. In another embodiment, the processing chamber further
comprises a strainer (e.g., filter or a mesh screen) positioned to
strain suspension of biological material entering the processing
chamber from the preprocessing chamber. In another embodiment, the
processing chamber communicates with a cartridge port configured
such that when vacuum is applied to the cartridge port, liquid in
the preprocessing chamber is pulled into the processing chamber. In
another embodiment, the cartridge further comprises a vacuum trap
fluidically connected with and positioned between the cartridge
port with the processing chamber. In another embodiment, the
processing chamber comprises a drain section and a more narrow slot
section and wherein the processing chamber comprises a slanted
floor configured to direct fluid in the drain section toward the
slot section. In another embodiment, the processing chamber
comprises a depression for collecting a suspension of biological
material. In another embodiment, the processing chamber
communicates with a cartridge port configured to introduce fluids
into the processing chamber. In another embodiment, the processing
chamber comprises a cover comprising a port that communicates
through a fluidic channel with the preprocessing chamber. In
another embodiment, the preprocessing chamber comprises a cover
comprising a seal (e.g. a friable seal, or septum) that, when
removed or opened (e.g., punctured), allows access to the
processing chamber. In another embodiment, the processing chamber
comprises a cover comprising a seal (e.g. a friable seal, or
septum) that, when removed or opened (e.g., punctured), allows
access to the processing chamber. In another embodiment, the
cartridge further comprises: one or more waste chambers fluidically
connected with the processing chamber. In another embodiment, the
cartridge further comprises an identifier comprising information
about the cartridge and/or its use (e.g., a barcode, an RFID, an EE
PROM), and wherein the instrument comprises a reader for reading
information in the identifier. In another embodiment, the one or
more sources of liquids and/or gasses are comprised in a fluidic
subsystem.
[0065] In another aspect provided herein is a method comprising:
(a) providing a system as disclosed herein, wherein the
preprocessing chamber comprises a tissue sample; (b) disrupting the
tissue sample by using the actuator to actuate the tissue disrupter
to produce a suspension of biological material; and (c) using the
fluidic subsystem to move the suspension of biological material
from the preprocessing chamber into the processing chamber. In one
embodiment, the method further comprises: removing the suspension
of biological material from the processing chamber. In another
embodiment, the prepossessing chamber further comprises one or more
enzymes for digesting extracellular matrix. In another embodiment,
the prepossessing chamber further comprises one or more detergents
for lysing cell membranes. In another embodiment, the prepossessing
chamber further comprises liquid having a viscosity that slows the
rate of degradation of RNA or other biomolecules during or after
tissue disruption. In another embodiment, disrupting comprises
positioning a disruption surface of the head a defined distance
from a bottom surface of the preprocessing chamber and rotating the
head to disrupt tissue in the preprocessing chamber. In another
embodiment, disrupting comprises positioning a disruption surface
of the head with respect to a bottom surface of the preprocessing
chamber at a plurality of different gap distances and, at each gap
distance, rotating the head. In another embodiment, at at least one
gap distance at least some portion of the disruption head contacts
some portion of the bottom surface. In another embodiment, the
widest gap distance between a flat portion of the head surface and
flat portion of the bottom of the chamber is no more than any of 6
mm, 5 mm 4 mm, 3 mm, 2 mm, 1 mm, 500 .mu.m, 250 .mu.m, 100 .mu.m,
75 .mu.m, 50 .mu.m, 25 .mu.m, 20 .mu.m, 15 .mu.m, 10 .mu.m, 5
.mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1 .mu.m. In another
embodiment, the plurality of gap distances between a flat portion
of the head surface and flat portion of the bottom of the chamber
is any of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the largest gap distance
is no is no more than any of 6 mm, 5 mm 4 mm, 3 mm, 2 mm, 1 mm, 500
.mu.m, 250 .mu.m, 100 .mu.m, 75 .mu.m, 50 .mu.m, 25 .mu.m, 20
.mu.m, 15 .mu.m, 10 .mu.m, 5 .mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1
um. In another embodiment, the method comprises: disrupting tissue
with the tissue disruptor; incubating the disrupted tissue with at
least one enzyme that digests extracellular matrix; and disrupting
the incubated tissue with the tissue disruptor. In another
embodiment, the fluidic subsystem applies a vacuum to a cartridge
port communicating with the processing chamber to move the
suspension of biological material. In another embodiment, the
cartridge further comprises a strainer and the suspension of
biological material entering the processing chamber is strained to
remove particulate matter, e.g., clumps of cells or nuclei or
subcellular biomolecules. In another embodiment, the method further
comprises, after moving the suspension of biological material,
using the fluidics subsystem to introduce a liquid into the
preprocessing chamber through a cartridge port and then using the
fluidics subsystem to move the liquid into the processing chamber.
In another embodiment, the method further comprises using the
fluidics subsystem to introduce one or more liquids comprising one
or more reagents through a cartridge port into the processing
chamber. In another embodiment, the reagent comprises an enzyme or
a particle comprising a binding agent (e.g., a binding agent
directed against a target on a cell surface or a surface of a
nucleus or surface of a virus, or other biological target). In
another embodiment, the tissue comprises a target cell and the
method further comprises: contacting the suspension of biological
material in the processing chamber with solid particles comprising
binding agents that bind to the target cells and sequester bound
target cells within the suspension of biological material. In
another embodiment, the method further comprises separating the
bound target cells from the suspension. In another embodiment, the
tissue is tumor tissue and the target cells are tumor infiltrating
lymphocytes. In another embodiment, the target cells are stem cells
or partially differentiated cells. In another embodiment, the
method further comprises: determining the expression of one or more
genes in cells, nuclei, mitochondria or other organelles of the
suspension of biological material. In another embodiment, the one
or more genes is a panel comprising a plurality of genes. In
another embodiment, the panel comprises genes distinguishing a
target cell type, e.g., hepatocytes, neurons, kidney glomerulus
parietal cell, cardiomyocytes. In another embodiment, the panel
comprises genes distinguishing a CRISPR modified target cell. In
another embodiment, the panel comprises genes that are
differentially expressed when cells experience stress, e.g.,
anoikis. In another embodiment, the method comprises preparing a
suspension of biological material on each of a plurality of tissue
samples under different tissue disruption conditions, and
identifying conditions that produce cells or nuclei having a gene
expression profile closest to or furthest away from that of cells
in the pre-disrupted tissue sample. In another embodiment, the
panel comprises one or more housekeeping genes, e.g., a gene
constitutively expressed at a relatively constant level in cells
regardless of cellular stress states, e.g., Actb, gapdh.
[0066] In another aspect provided herein is a cartridge comprising:
(i) a preprocessing chamber comprising: (1) an opening and,
positioned in the opening, a tissue disruptor configured for
mechanical disruption of tissue, and (2) a preprocessing chamber
port; and (ii) a processing chamber comprising a processing chamber
port communicating with the preprocessing chamber port through a
fluid line, and (iii) a cartridge port that communicates with the
processing chamber, wherein a vacuum applied to the cartridge port
pulls material from the preprocessing chamber into the processing
chamber. In one embodiment, the cartridge port communicates with
the processing chamber directly or through a vacuum trap.
[0067] In another aspect provided herein is a cartridge comprising:
(i) a preprocessing chamber comprising an opening and, positioned
in the opening, a tissue disruptor configured for mechanical
disruption of tissue; (ii) a strain chamber comprising a strainer,
wherein the strain chamber communicates with the preprocessing
chamber; (iii) a first processing chamber communicating with the
strain chamber; (iv) an optional second processing chamber
communicating with the first processing chamber; (v) one or more
cartridge ports communicating with the processing chamber and the
second processing chamber if present.
[0068] In one embodiment, the cartridge further comprises: one or
more waste chambers communicating with the first processing chamber
and second processing chamber when present. In one embodiment, the
first processing chamber comprises an element (e.g., a nozzle)
configured to produce a hanging drop of liquid from the strain
chamber.
[0069] In another aspect provided herein is a method of creating a
microtissue comprising: (a) providing a cartridge of as described
herein comprising a tissue; (b) disrupting the tissue with the
tissue disruptor to produce a cell suspension; (c) straining the
cell suspension with the strainer to produce strained cell
suspension; and (d) forming a hanging drop from the strained cell
suspension using the element. In one embodiment, the microtissue is
an organoid. In another embodiment, the method further comprises:
after forming the hanging drop, adding a liquid or gas to the
processing chamber to support survival of the cells in the hanging
drop.
[0070] In another aspect provided herein is a method of creating a
microtissue comprising: (a) providing a cartridge of as described
herein comprising a tissue; (b) disrupting the tissue with the
tissue disruptor to produce a cell suspension; (c) straining the
cell suspension with the strainer to produce strained cell
suspension; (d) selecting stem cells from strained cell suspension;
and (e) removing or growing the selected stem cells in the
cartridge.
[0071] In another aspect provided herein is a method of creating a
microtissue comprising: (a) providing a cartridge of as described
herein comprising a tissue; (b) disrupting the tissue with the
tissue disruptor to produce a cell suspension; (c) straining the
cell suspension with the strainer to produce strained cell
suspension; (d) differentiating cells from strained cell suspension
into stem cells; and (e) growing or removing the differentiated
stem cells in the cartridge.
[0072] In another aspect provided herein is a system comprising:
(a) an instrument comprising: (i) one or more cartridge interfaces,
each configured to engage a cartridge and comprising one or more
fluid ports; (ii) a module comprising an actuator to actuate a
tissue disruptor in a cartridge engaged with a cartridge interface
(e.g., a mechanical, pneumatic, electromagnetic, or hydraulic
actuator); and (b) one or more cartridges, each engaged with one of
the cartridge interfaces, wherein each cartridge comprises: (i) a
preprocessing chamber comprising an opening and, positioned in the
opening, a tissue disruptor configured for mechanical disruption of
tissue. In one embodiment the cartridge does not include any
chambers other than the preprocessing chamber.
[0073] In another aspect provided herein is a cartridge comprising:
(i) a preprocessing chamber comprising an opening and, positioned
in the opening, a tissue disruptor configured for mechanical
disruption of tissue.
[0074] In another aspect provided herein is a tissue disruptor
comprising: (i) a sheath, (ii) a plunger configured to move
slidably within the sheath and comprising a coupler to engage the
actuator and a head for disrupting tissue, and (iii) a bias
mechanism, e.g., spring, that biases the plunger toward a retracted
position, i.e., wherein actuation is required to actuate the
plunger to a forward position. In one embodiment, the sheath
comprises a seater element adapted to seat the tissue disruptor on
a seat. In another embodiment, the seater element comprises a
flange adapted to sit on an annular ring. In another embodiment,
the seater element comprises one or more tabs adapted to sit in one
or more slots.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0076] FIG. 1 shows a Sample Processing System that processes
specimens or tissue specimens into biocomponents such as single
cells or nuclei for bioanalysis.
[0077] FIG. 2 shows a Tissue Processing System that processes
tissue specimens into biocomponents such as single cells or nuclei
or other for bioanalysis.
[0078] FIG. 3 shows a high level overview of the workflow for a
Singulator System to generate for example single cell or nuclei or
biomolecules from a specimen or tissue specimen.
[0079] FIG. 4 shows an overview of an embodiment of the Singulator
System and some exemplary modules. Tissue specimens or other
specimens processed into single cells, nuclei, nucleic acids,
single-cell libraries, microtissues, organoids and other
biologicals through the use of one or more cartridges and one or
more of the Physical Dissociation Subsystem, Enzymatic and Chemical
Dissociation Subsystem, Measurement Subsystem, Fluidic Subsystem,
Control Subsystem, or a Magnetic Module.
[0080] FIG. 5 shows the overall design concept of the Cell
Singulation module for a prototype showing functional modules and a
few example modalities of mechanical disruption and example
enyzmatic formulation to dissociate solid tissue specimens into
single cells, nuclei, and other biomolecules.
[0081] FIG. 6 shows an example of a Single-Sample Singulation
System with mechanical disruption in a single cartridge with a bank
of enzymes and reagents located in the instrument to dissociate
solid tissue specimens into single cells, nuclei, and other
biomolecules.
[0082] FIG. 7 shows another example of a Single-Sample Singulation
System with mechanical disruption in a single cartridge with a bank
of enzymes and reagents located separately from the instrument in a
reagent module.
[0083] FIGS. 8A and B shows an example of a reagent module for the
Single-Sample Singulation System to dissociate solid tissue
specimens into single cells, nuclei, and other biomolecules.
[0084] FIG. 9 shows the front of an example of the Single-Sample
Singulation System to dissociate solid tissue specimens into single
cells, nuclei, and other biomolecules using a cartridge.
[0085] FIG. 10 shows the back of an example of the Single-Sample
Singulation System.
[0086] FIG. 11 shows an example of a two sample Singulation System
to dissociate solid tissue specimens into single cells, nuclei, and
other biomolecules using a two cartridges.
[0087] FIG. 12 shows an example of a cartridge with preprocessing,
processing, and vacuum trap chambers for processing solid tissue
specimens into single cells, nuclei, and other biomolecules.
[0088] FIGS. 13A-C show an example of a cap with a cartridge with a
preprocessing, processing, and vacuum trap chambers for processing
solid tissue specimens into single cells, nuclei, and other
biomolecules and details of the assembly of the cap.
[0089] FIGS. 14A-B shows an example of a tissue disruptor with a
feature designed to center the head of the disruptor in a
preprocessing chamber and set the bottom gap and side gaps between
the disruptor head and the wall of the preprocessing chamber.
[0090] FIGS. 15A-D show a port cover with low durometer over a port
secured by a port cover retaining cylinder, or a crimp, or a heat
staked port cover retaining cylinder.
[0091] FIGS. 16A-E shows a cap engaging with a rotor motor adaptor
and with a cartridge with a preprocessing, processing, and vacuum
trap chambers for processing solid tissue specimens into single
cells, nuclei, and other biomolecules.
[0092] FIG. 17 shows the reagent module with reagents loaded in an
exemplary setup.
[0093] FIG. 18A shows an example of a single cell suspension of
mouse kidney dissociated on the Singulator system. FIG. 18B shows
an example of a nuclei suspension of mouse kidney dissociated on
the Singulator system.
[0094] FIG. 19A shows an example of a vertical cartridge that
integrates processing of tissue with the formation of an organoid
by the hanging drop method and FIG. 19B is an illustration of the
backside of the exemplary vertical cartridge.
[0095] FIG. 20 shows a closeup of a vertical cartridge with a
hanging droplet being formed on a noozle.
[0096] FIG. 21 shows a panel of genes useful in measuring stress
induced gene expression changes.
DETAILED DESCRIPTION OF THE INVENTION
[0097] NGS, mass spectrometry, FACS, and other modern
high-throughput analysis systems have revolutionized life and
medical sciences. The progression of information has been from the
gross level of organism, to tissue, and now to single cell
analysis. Single cell analysis of genomic, proteomic including
protein expression, carbohydrate, lipid, and metabolism of
individual cells is providing fundamental scientific knowledge and
revolutionizing research and clinical capabilities.
[0098] Specimen: The term "specimen," as used herein, refers to an
in vitro cell, cell culture, virus, bacterial cell, fungal cell,
plant cell, bodily sample, or tissue sample that contains genetic
material. In certain embodiments, the genetic material of the
specimen comprises RNA. In other embodiments, the genetic material
of the specimen is DNA, or both RNA and DNA. In certain embodiments
the genetic material is modified. In certain embodiments, a tissue
specimen includes a cell isolated from a subject. A subject
includes any organism from which a specimen can be isolated.
Non-limiting examples of organisms include prokaryotes, eukaryotes
or archaebacteria, including bacteria, fungi, animals, plants, or
protists. The animal, for example, can be a mammal or a non-mammal.
The mammal can be, for example, a rabbit, dog, pig, cow, horse,
human, or a rodent such as a mouse or rat. In particular aspects,
the tissue specimen is a human tissue sample. The tissue specimen
can be liquid, for example, a blood sample, red blood cells, white
blood cells, platelets, plasma, serum. The specimen, in other
non-limiting embodiments, can be saliva, a cheek, throat, or nasal
swab, a fine needle aspirate, a tissue print, cerebral spinal
fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal
fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid,
tears, semen, seminal fluid, vaginal fluids, pulmonary effusion,
serosal fluid, organs, bronchio-alveolar lavage, tumors, frozen
cells, or constituents or components of in vitro cell cultures. In
other aspects, the tissue specimen is a solid tissue sample or a
frozen tissue sample or a biopsy sample such as a fine needle
aspirate or a core biopsy or a resection or other clinical or
veterinary specimen. In still further aspects, the specimen
comprises a virus, archae, bacteria, or fungus. The specimen can be
an ex vivo tissue or sample or a specimen obtained by laser capture
microdissection. The specimen can be a fixed specimen, including as
set forth by U.S. Published Patent Application No. 2003/0170617
filed Jan. 28, 2003, or a FFPE specimen.
[0099] In some embodiments, the single cells can be analyzed
further for biomolecules including one or more polynucleotides or
polypeptides or other macromolecules. In some embodiments, the
polynucleotides can include a single-stranded or double-stranded
polynucleotide. In some embodiments, the polypeptide can include an
enzyme, antigen, hormone or antibody. In some embodiments, the one
or more biomolecules can include RNA, mRNA, cDNA, DNA, genomic DNA,
microRNA, long noncoding RNA, ribosomal RNA, transfer RNA,
chloroplast DNA, mitochondrial DNA, or other nucleic acids
including modified nucleic acids and complexes of nucleic acids
with proteins or other macromolecules.
[0100] It will be readily apparent to one of ordinary skill in the
art that the embodiments and implementations are not necessarily
inclusive or exclusive of each other and may be combined in any
manner that is non-conflicting and otherwise possible, whether they
be presented in association with a same, or a different, embodiment
or implementation. The description of one embodiment or
implementation is not intended to be limiting with respect to other
embodiments and/or implementations. Also, any one or more function,
step, operation, or technique described elsewhere in this
specification may, in alternative implementations, be combined with
any one or more function, step, operation, or technique described
in the summary. Thus, the above embodiment and implementations are
illustrative rather than limiting.
[0101] One version of tissue processing system is decribed in
International patent application PCT/US2017/063811 filed Nov. 29,
2017 (WO 2018/102471) (Jovanovich, Chear, McIntosh, Pereira, and
Zaugg, "Method and Apparatus for Processing Tissue Samples"),
incorporated herein in its entirely for all purposes.
[0102] FIG. 1 shows a Sample Processing System 50 that can input
specimen 101 and process them to produce biologicals such as single
cells 1000 or nuclei 1050, microtissues 6001, organoids 6002, or
other biocomponents comprised of subcellular components 1060, and
biomolecules 1070 such as macromolecules 1071 and nucleic acids
1072, comprised of DNA 1073 and RNA 1074; proteins 1075;
carbohydrates 1076; lipids 1077; biomolecules 1070 with multiple
types of macromolecules 1071; metabolites 1078; and other
biological components, including natural products 1079 for
bioanalysis.
[0103] FIG. 2 shows a Tissue Processing System 80 that can input
tissue specimens 120 and other specimens 101 and process them to
produce biologicals such as single cells 1000 or nuclei 1050,
microtissues 6001, organoids 6002, or other biocomponents comprised
of subcellular components 1060, and biomolecules 1070 such as
macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073
and RNA 1074; proteins 1075; carbohydrates 1076; lipids 1077;
biomolecules 1070 with multiple types of macromolecules 1071;
metabolites 1078; and other biological components, including
natural products 1079 for bioanalysis.
[0104] Referring to FIG. 3, the Singulation System 100 accepts one
or more specimens 101 or tissue specimens 120 and processes them to
produce biologicals such as single cells 1000 or nuclei 1050,
microtissues 6001, organoids 6002, or other biocomponents comprised
of subcellular components 1060, and biomolecules 1070 such as
macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073
and RNA 1074 and single cell libraries 1200 for bioanalysis.
[0105] Referring to FIG. 4, in some embodiments, the Singulation
System 100 processing is performed in cartridge(s) 200 in the
system. Tissue specimens 120 or other specimens 101 are converted
to single cells 1000, nuclei 1050, nucleic acids 1072, single cell
libraries 1200, single nuclei libraries 1250, bulk libraries 1210,
or other biocomponents comprised of subcellular components 1060, or
biomolecules 1070 such as macromolecules 1071 and nucleic acids
1072, comprised of DNA 1073 and RNA 1074, or microtissues 6001, or
organoids 6002 through the use of one or more cartridges 200 with
one or more of the Physical Dissociation Subsystem 300, the
Enzymatic and Chemical Dissociation Subsystem 400, the Measurement
Subsystem 500, the Fluidic Subsystem 600, the Control Subsystem
700, Temperature Subsystem 1475, and the Magnetic Module 900.
[0106] The Physical Dissociation Subsystem 300 (which can include a
preprocessing chamber, a tissue disruptor and an actuator) can
perform physical disruption by passing the specimen through
orifices, grinding, rotating a rotor with or without features to
dissociate tissue, moving a head with or without features to
dissociate tissue, forcing tissue through filters or screens or
mesh or strainers, moving a pestle or Dounce like element,
sonication, blending, homogenization, bead beating, pressure, and
other methods known to one skilled in the art to physically disrupt
tissue to help produce single cells or nuclei.
[0107] The Enzymatic and Chemical Dissociation Subsystem 400 (which
can include or use a source of fluid (e.g., comprising one or more
enzymes or chemicals) and portions of the fluidic subsystem and
cartdrige interface that deliver liquids to a preprocessing or
other chambers) can perform enzymatic disruption by adding
formulations of a reagents or mixture of components comprised of
but not limited to collagenases (e.g., collagenases type I, II,
Ill, IV, and others), elastase, trypsin, papain, hyaluronidase,
chymotrypsin, neutral protease, clostripain, caseinase, neutral
protease (Dispase.RTM.), DNAse, protease XIV, RNase inhibitors,
DNAse inhibitors, or other enzymes, biochemicals, or chemicals such
as EDTA, EGTA, protease inhibitors, buffers, acids, or base.
[0108] In another aspect, the Enzymatic and Chemical Dissociation
Subsystem 400 can perform chemical disruption or chemical and
enzymatic disruption is by adding formulations of chemicals that
might disrupt tissue or cellular integrity such as Triton X-100,
Tween, Nonident P40, other surfactants or detergents, digitonin, or
biomolecules or chemicals that can dissociate tissue into cells or
produce nuclei or other organelles directly from tissues or from
single cell 1000 suspensions. Many different nuclei isolation
solutions 412 have been developed, including NST buffer (146 mM
NaCl, 10 mM Tris base at pH 7.8, 1 mM CaCl.sub.2, 21 mM MgCl.sub.2,
0.05% BSA, 0.2% Nonidet P-40) (L. Martelotto, T Baslan, J, Kendall,
F. C Geyer, K. A Burke, L. Spraggon, S. Piscuoglio, K. Chadalavada,
G. Nanjangud, C. Ng, P. Moody, S. D'Italia, L. Rodgers, H. Cox, A.
da Cruz Paula, A. Stepansky, M. Schizas, H. Y. Wen, T. A King, L.
Norton, B. Weigelt, J. B Hicks, and J. S. Reis-Filho. Whole-genome
single-cell copy number profiling from formalin-fixed
paraffin-embedded samples. Nat Med. 2017 Mar. 23(3): 376-385.
doi:10.1038/nm.4279.) or Homogenization buffer (10 mM Tris pH 8.0,
250 mM sucrose, 25 mM KCl, 5 mM MgCl.sub.2, 0.1% Triton-X 100,
(v/v), 0.4 U/.mu.L RNasin Plus RNase inhibitor (Promega), lx
protease inhibitor, 0.2 U/.mu.L Superasin (ThermoFisher), 10 ng/mL
Hoechst 33342, and 0.1 .mu.M DTT) (Krishnaswami S R, Grindberg R V,
Novotny M, Venepally P, Lacar B, Bhutani K, Linker S B, Pham S,
Erwin J A, Miller J A, Hodge R, McCarthy JK, Kelder M, McCorrison
J, Aevermann BD, Fuertes FD, Scheuermann RH, Lee J, Lein E S,
Schork N, McConnell M J, Gage F H, Lasken R S. Using single nuclei
for RNA-seq to capture the transcriptome of postmortem neurons. Nat
Protoc. 2016 Mar.; 11(3):499-524. doi: 10.1038/nprot.2016.015.
PMID: 26890679.), or nuclear homogenization buffer (10 mM
HEPES-KOH, pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10% glycerol,
0.15 mM spermine, 0.5 mM spermidine, 10 mM NaF, 1 mM orthovanadate,
1 mM PMSF, 0.5 mM DTT, and 1X protease inhibitor cocktail (Sigma))
(Ling G, Waxman D J. Isolation of nuclei for use in genome-wide
DNase hypersensitivity assays to probe chromatin structure. Methods
Mol Biol. 2013; 977:13-9. doi: 10.1007/978-1-62703-284-1_2. PubMed
PMID: 23436350; PubMed Central PMCID: PMC3815455.) or 0.1x Lysis
Buffer (1 mM TrisHCl, pH7.4, 1 mM NaCl, 0.3 mM MgCl.sub.2, 0.01%
Tween-20, 0.01% Nonldent P40, 0.001% digitonin, 0.1% bovine serum
albumin) (Demonstrated Protocol-Nuclei Isolation from Mouse Brain
Tissue for Single Cell ATAC Sequencing, Rev A, 10x Genomics) or
1.times.NIB: (10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM KCl, 2.5 mM
EDTA, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT.
(S. Sikorskaite, M.-L. Rajamaki, D. Baniulis, V. Stanys and J. PT
Valkonen. Protocol: Optimised methodology for isolation of nuclei
from leaves of species in the Solanaceae and Rosaceae families.
Plant Methods 2013, 9:31
http://www.plantmethods.com/content/9/1/31).
[0109] Similarly many different nuclei storage solutions 413 have
been developed including Nuclei Wash and Resuspension Buffer
(1.times.PBS with 1% BSA and 0.2 u/mL RNase Inhibitor
(Sigma-Aldrich 3335399001)) (Demonstrated Protocol-Isolation of
Nuclei for Single Cell RNA Sequencing, Rev B, 10x Genomics), or
nuclear storage buffer (20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 0.5 mM
EDTA, 50% (v/v) glycerol, 1 mM DTT, and 0.1 mM PMSF) (Ling G,
Waxman D J. Isolation of nuclei for use in genome-wide DNase
hypersensitivity assays to probe chromatin structure. Methods Mol
Biol. 2013;977:13-9. doi: 10.1007/978-1-62703-284-1_2. PubMed PMID:
23436350; PubMed Central PMCID: PMC3815455.) or nuclear storage
buffer (20% glycerol, 20 mM HEPES-KOH (pH 7.2), 5 mM MgCl.sub.2, 1
mM DTT) (S. Sikorskaite, M.-L. Rajamaki, D. Baniulis, V. Stanys and
J. P T Valkonen. Protocol: Optimised methodology for isolation of
nuclei from leaves of species in the Solanaceae and Rosaceae
families. Plant Methods 2013, 9:31
http://www.plantmethods.com/content/9/1/31) or NSB (166.6 mM
sucrose, 5 mM MgCl.sub.2, 10 mM Tris buffer, pH 8.0) (Krishnaswami
S R, Grindberg R V, Novotny M, Venepally P, Lacar B, Bhutani K,
Linker S B, Pham S, Erwin J A, Miller J A, Hodge R, McCarthy J K,
Kelder M, McCorrison J, Aevermann B D, Fuertes F D, Scheuermann R
H, Lee J, Lein E S, Schork N, McConnell M J, Gage F H, Lasken R S.
Using single nuclei for RNA-seq to capture the transcriptome of
postmortem neurons. Nat Protoc. 2016 Mar.; 11(3):499-524. doi:
10.1038/nprot.2016.015. PMID: 26890679.).
[0110] In other embodiments, different reagents or mixtures of
reagents are applied sequentially to dissociate the biological
sample or specimen into single cells or nuclei. The physical and
enzymatic/chemical dissociation and other subsystems can be
separate from each other, or they can be co-located (e.g., acting
upon the sample simultaneously or sequentially). The preprocessing,
strain, and processing chambers can be separate from each other, or
they can be co-located (e.g., acting upon the sample simultaneously
or sequentially).
[0111] In some embodiments, the amount of dissociation is monitored
at intervals during the dissociation or at the endpoint, and in
some instances the viability is determined during processing using
a Measurement Subsystem 500. The Measurement Subsystem 500 can be
an optical imaging device to image cells using brightfield, phase
contrast, fluorescence, chemiluminescence, near-field, Raman, or
other optical readouts, or an optical measurement, or an electrical
measurement, such as an impedance measurement of the change in
conductivity, when a cell passes through a sensor, or thermal, or
other types of measurement. In other embodiments Measurement
Subsystem 500 can be a mass spectrometer, mass cytometer, or other
system that determines mass.
[0112] The addition and movement of fluids can be performed by a
Fluidic Subsystem 600. The Fluidic Subsystem 600 can use pumps,
such as syringe pumps, piezopumps, electroosmotic pumps, peristalic
pumps, on-cartridge pumps and valves, micropumps, pressure,
pneumatics, or other components well known to one skilled in the
art.
[0113] The Singulation System 100 can be controlled by software in
a Control Subsystem 700 which can be comprised of a user interface
740 through a monitor, embedded display, or a touch screen 730. In
some instances the Control Subsytem 700 can include interfaces to
laboratory information management systems, other instruments,
analysis software, display software, databases, email, and other
applications. The Control Subsystem 700 can include control
software 725 and scripts that control the operation and in some
embodiments the scripts can be revised, created, or edited by the
operator.
[0114] The Singulation System 100 can have temperature subsystem
1475 for temperature regulation that can set the temperature of
various parts of the system such as at reagent storage, or in
fluidic lines, or in cartridge 200. The temperature subsystem 1475
can use heating and or cooling from devices comprised of resistive
heaters, Peltiers, circulating fluids, or other methods well known
to one skilled in the art, with a temperature sensing element, such
as a thermistor, thermocouple, thermoresponse color change, etc.,
and a temperature control board.
[0115] In another aspect provided herein is a device for the
dissociation of a biological sample, the device comprising: (i) a
biological sample or specimen 101; (ii) a cartridge 200 capable of
dissociating tissue; (iii) an instrument to operate the cartridge
200 and provide fluids as needed (iv) a measurement module 500 such
as an optical imaging to measure titer, clumping, and/or viability,
or realtime PCR, (v) exchange of dissociation solution for buffer
or growth media at the desired titer, and (vi) output vessels such
as a chamber in the cartridge, 8 well strip tubes, microtiter
plates, Eppendorf tubes, nanowells, or other vessels capable of
receiving cell suspensions or an organoid 6002 or microtissue
6001.
[0116] In another aspect provided herein is a device for the
dissociation of a biological sample and the production of
single-cell 1000 or nuclei 1050 suspensions or matched bulk nucleic
acids 1010 or single cell libraries 1200 or matched bulk libraries
1210, the device comprising: (i) a chamber or area to input a
biological sample or specimen; (ii) a cartridge capable of
dissociating tissue or specimen; (iii) an instrument to operate the
cartridge and provide fluids as needed (iv) a measurement module
such as an optical imaging to measure titer, clumping, and/or
viability, or the quantity of one or more biomolecules 1070, (v)
exchange of dissociation solution for buffer or growth media at the
desired titer, (vi) the production of single-cell 1000 or nuclei
1050 suspensions or single cell libraries 1200, and matched bulk
nucleic acid libraries 1210, in output vessels such as 8 well strip
tubes, microtiter plates, Eppendorf tubes, a chamber in the
cartridge, or other vessels capable of receiving cell
suspensions.
[0117] Still referring to FIG. 4, a Magnetic Processing module 900
can use magnetic processing of magnetic and paramagnetic particles
or surfaces or beads, referred to as beads, to separate single
cells 1000, or cell types or nuclei 1050, or other biocomponents
comprised of subcellular components 1060, and biomolecules 1070
such as macromolecules 1071 and nucleic acids 1072, comprised of
DNA 1073 and RNA 1074; proteins 1075; carbohydrates 1076; lipids
1077; biomolecules 1070 with multiple types of macromolecules 1071;
metabolites 1078; and other biological components, including
natural products 1079 for bioanalysis. The magnetic processing
module can introduce a magnetic field into parts of the cartridge,
e.g., a processing chamber or other chamber or part of a chamber.
This field can be used exert a magnetic force on magnetic and
paramagnetic materials in the field, such as particles, such as
beads, such as surfaces. Such particles can be sequestered from
fluids in the chamber and, ultimately, separated from the fluids.
In some embodiments the beads have a surface chemistry that
facilitates the purification of the biologicals in conjunction with
the chemical conditions. In other embodiments the bead have
affinity molecules comprised of antibodies, aptamers, biomolecules,
etc. that specifically purify certain biologicals such as cell
types, organelles, nucleic acids 1072, nuclei 1050, or other
components of tissue or samples.
[0118] In another aspect provided herein is a device for the
dissociation and single-cell or single nuclei library preparation
of a biological sample, the device comprising: (i) a chamber or
area to input a biological sample or specimen; (ii) a cartridge 200
capable of dissociating tissue specimens 120 into single-cells 1000
or single nuclei 1050 and then producing single-cell libraries 1200
or single-nuclei libraries 1250; (iii) an instrument to operate the
cartridge 200 and provide fluids as needed (iv) a measurement
subsystem 500 such as an optical imaging to measure titer,
clumping, and/or viability, (v) exchange of dissociation solution
for buffer at the desired titer, (vi) a magnetic processing or
other processing chamber or tubing to perform magnetic separations,
normalizations, purifications, and other magnetic processes, for
example, to purify nucleic acids, couple enyzmatic reactions such
as library preparation reactions, and other processes including
producing single-cells or nuclei in isolation, such as nanodrops,
nanoboluses, or physical separation or solutions including agarose,
polyethylene gycol, and other chemicals and materials that slow
diffusion, (vii) output vessels such as 8 well strip tubes,
microtiter plates, Eppendorf tubes, a chamber in the cartridge, or
other vessels capable of receiving cell suspensions including
nanowells.
[0119] In another embodiment, herein is a device and method for the
dissociation of tissue into single cells which are then used to
form microtissues 6001 or organoids 6002 on the cartridge 200 or
the single-cells 1000 are used off the cartridge 200 to create
microtissues 6001 or organoids 6002.
[0120] The basic elements of the Singulation System 100 can be
configured in multiple ways depending on the specimen(s) 101 and
analytes to be analyzed. In the following examples, a few of the
numerous configurations are described in detail but in no way is
the invention limited to these configurations as will be obvious to
one skilled in the art. The Singulation System 100 can accommodate
many different types of specimens 101, comprised of fresh tissue;
snap-frozen tissue; microtome slices (cryo, laser or vibrating) of
tissue; fixed tissue; FFPE; bulk material obtained by surgical
excision, biopsies, fine needle aspirates; samples from surfaces,
and other matrices.
[0121] There is a need to fill gap in the single-cell sample
preparation for microtissues 6001 or organoids 6002 by starting the
workflow at processing raw solid tissues into single-cell 1000
suspensions. The instant disclosure teaches how to produce a system
that processes tissue specimens 120 and other samples into single
cell 1000 suspensions nd then form microtissues 6001 or organoids
6002 with little or no intervention by the operator once the
process is started. This requires adapting to the widely varying
starting types of tissue, with different requirements depending on
the tissue, species, age, and state. In some embodiments, the cells
are used to isolate tumor infiltrating lymphocytes which can be
characterized by sequencing or flow cytometery, or cultured with
lymphokines such as interleukin-1 to produce therapeutic tumor
infiltrating lymphocytes. The therapeutic tumor infiltrating
lymphocytes can then be infused into the patient to lyse tumor
cells and combat disease progression.
[0122] In the instant invention, many embodiments are possible and
are incorporated by reference from patent application
PCT/US2017/063811 filed Nov. 29, 2017 (Jovanovich, Chear, McIntosh,
Pereira, and Zaugg, "Method and Apparatus for Processing Tissue
Samples") and from provisional patent application 62/679,466 filed
Jun. 1, 2018 (Jovanovich, "Method and Apparatus for Processing
Tissue Samples); the contents of all are incorporated herein in
their entirety as well as the number system used therein. Systems
with increasing capabilites can be developed as a series of
embodiments, six are described: two embodiment as a Single Sample
Singulator System 2000, one as a Two Sample Singulator System 2200,
a Four Sample Singulator System 2400, an Enhanced Singulator System
2500, and the Single Librarian 3000 embodiments.
[0123] This disclosure describes how to automate, integrate, and
importantly standardize the complete process to create single-cell
1000 and then produce microtissues 6001 or organoids 6002 in a
single Singulator System 100 system embodiment. The Singulator
System 100 will greatly enable basic researchers, students, and
translational researchers as well as clinicians and others with its
ease of use and high performance.
[0124] Single-Use Cartridge Designs.
[0125] Cartridges 200 can be used to process tissue into
single-cell 1000 suspensions or nuclei 1050 and are preferrablely
single-use. The major workflow steps to produce single-cell
suspensions 1000 for the production of microtissues 6001 or
organoids 6002 is to first mechanically disrupt solid tissue by
enzymatic dissociation, and straining to remove clumps.
[0126] Referring to FIG. 5, cartridge 200 will input specimen 101
and output viable singulated cells 1000 that are used to create
microtissues 6001 or organoids 6002 or in some embodiments, as
illustrated in FIGS. 19 and 20 by a hanging droplet 6200 in the
cartridge 200. It is desirable that disposable cartridge 200
process multiple types of samples with mechanical disruption and
enzymatic or chemical dissociation according to the tissue type and
condition. The cartridge 200 can be designed to process tissue as
quickly and as gently as possible, not expose the operator to the
tissue being processed, and be manufacturable at low cost. Multiple
mechanical methods may be needed to accommodate the wide range of
tissues and their individual requirements: designs are shown that
can be readily adapted to multiple different mechanical disruption
methods comprising variable orifice 490, grinding with rotating
plungers 336, pestles 361, and straining and filtering using a
plunger 362 as well as other mechanical methods without
limitation.
[0127] Cartridges 200 can be designed for 3D printing, injection
molding in plastics with single or double pulls and low labor
assembly, or layered assembly of fluidic and other layers,
combinations of methods, and other methods well known to one
skilled in the art. Fluids can be delivered to cartridge 200 by a
syringe pump 2130 or can be preloaded onto cartridge 200 or many
combinations. In some embodiments, flexible tubing 493 can connect
chambers and creates simple pinch valves 491 to direct flow or can
be used to create a peristaltic pump. In other embodiments,
channels are created in the cartridge 200 and valves can be
incorporated such as pneumatic valves, or other valves.
[0128] Singulator System Embodiment
[0129] In one embodiment of the Sample Processing System 50 as a
Tissue Processing System 80, as shown in FIG. 2, the Singulator
System 100 can perform powerful integrated tissue-to-genomics or
sample-to-other answer (genomic, proteomic, metabolomic, or
epigenetic, multi-omics, etc.) analysis functionality for
scientists to simply and standardize the production and or analysis
of single-cell 1000 or nuclei 1050 suspensions, affinity purified
single cells 1100, affinity purified nuclei 1105 , nucleic acids
1072, and bulk libraries 1210 from solid or liquid tissues. As will
be obvious to one skilled in the art, the biological materials
produced such as single cells 1000, nuclei 1050, nucleic acids
1072, single cell libraries 1200, single nuclei libraries 1250,
bulk libraries 1210, or other biocomponents comprised of
subcellular components 1060, or biomolecules 1070 such as
macromolecules 1071 and nucleic acids 1072, comprised of DNA 1073
and RNA 1074, or microtissues 6001, or organoids 6002 can also be
used for many genomic, cell biology, proteomics, metabolomics, and
other analytical methods.
[0130] The Singulator System 100 can integrate the preparation of
biological materials from liquid or solid tissue(s) with
measurement subsystems 500 that perform an analysis selected from
one or more of: DNA or RNA sequencing, next generation DNA or RNA
sequencing, next next generation DNA or RNA sequencing of nucleic
acids and their adducts such as epigenetic modifications; nanopore
sequencing of nucleic acids and their adducts; single cell DNA
sequencing of nucleic acids and their adducts; single nuclei RNA
sequencing of nucleic acids and their adducts; PCR, digital droplet
PCR, qPCR, RT-qPCR; genomic analysis, gene expression analysis,
gene mapping, DNA fragment mapping; imaging including optical and
mass spectrometry imaging; DNA or RNA microarray analysis;
fluorescent, Raman, optical, mass spectrometery and other detection
modalities of nucleic acids acids and their adducts with and
without labels; proteomic analysis including fluorescent, Raman,
optical, mass spectrometery, protein sequencing, and other
detection modalities of proteins and peptides and their adducts and
modifications with and without labels; carbohydrate
characterization and profiling including sequencing, fluorescent,
Raman, optical, mass spectrometery, and other detection modalities
of carbohydrates and their adducts and other covalent polymers with
and without labels; lipid characterization and profiling including
sequencing, fluorescent, Raman, optical, mass spectrometery, and
other detection modalities of lipids and their adducts and other
covalent polymers with and without labels; flow cytometry;
characterization of cells and profiling including fluorescent,
Raman, optical, mass spectrometery, and other detection modalities
of cells and their adducts and other covalent polymers with and
without labels; metabolic profiling including sequencing,
fluorescent, Raman, optical, mass spectrometery, and other
detection modalities of metabolites and their adducts and other
covalent polymers with and without labels; functional analysis
including protein-protein interactions, protein-lipid interactions,
protein-DNA interactions, RNA-DNA interactions, and other
interactions between molecules derived from biological materials,
with and without labels; bioinformatic analysis of cells,
organelles, and biomolecules; and mass spectrometry and other
analytical methods.
[0131] In this preferred embodiment a Cell Singulation module 800
and a Magnetic Processing module 900 are integrated into a
Single-Sample Singulator System 2000 or into a Two-Sample
Singulator System 2200 or a Four-Sample Singulator System 2400 or
other Singulator system that processes more than four samples.
Mechanical and enzymatic dissociation is performed in single-use
cartridges 200 in one or more preprocessing chambers 440 to produce
single-cell suspension 1000 or nuclei suspensions 1200, nucleic
acids 1072, biomolecules 1070, subcellular components 1060, or
other products from pre-processing. The samples can then be
processed in the one or more processing chamber(s) 460 by optional
bead-based affinity purification of cell types by surface antigens
to produce affinity purified single-cell suspensions 1100 or
nuclear suspensions by nuclear antigens 1105 or nucleic acids 1072,
biomolecules 1070, subcellular components 1060 can be further
processed into purified mRNA, NGS libraries, or other sample types.
In some embodiments, one or more of the preprocessing 440 and
processing chambers 460 and strain chambers 450 and vacuum trap
chambers 468 and waste chambers 430 or other chambers can be
combined.
[0132] In a preferred embodiment, a Single-Sample Singulator System
2000 was designed with reagents 411 on-board or in a reagent module
1430 adjacent to the Single-Sample Singulator instrument 2050 and
with cartridges 200 incorporating one or more tissue-specific
mechanical disruption modalities to accommodate the wide diversity
of processing needs for tissue specimens 120. The system can input
raw, unprocessed tissue samples and output single-cells 1000 or
nuclei 1050 in suspension, ready for processing into single cell
NGS libraries off device or can process the single cells 1000 or
nuclei 1050 into bulk libraries on the system or perform analysis
of the processed tissues.
[0133] Example: A Single-Sample Singulation System to Create
Microtissues or Organoids.
[0134] The Singulator System 100 can mechanically disrupt tissue
and enzymatically dissociate the disrupted tissue in a cartridge
200 into single-cells 1000. As shown in the FIG. 5 in one
embodiment, a Cell Singulation module 800 or, as shown in FIG. 6, a
Single Sample Singulator System 2000 can combine the Physical
Dissociation Subsystem 300 and the Enyzmatic and Chemical
Dissociation Subsystem 400 to produce single-cell 1000 or nuclei
1050 suspensions. The instrument provides the mechanical motion and
fluidics to the cartridge which in turn mechanically and
enzymatically or chemically process the tissue into single cells
1000 or nuclei 1050. Multiple reagents 411 can be stored on the
instrument or reagent module 1430 with cooling as needed. The
single cell 1000 suspension can in turn be used to generate
microtissues 6001 or organoids 6002.
[0135] The Cell Singulation module 800 as shown conceptually in
FIG. 5 combines the mechanical disruption of specimen 101 on
cartridge 200, adds enzymatic or chemical dissolution solution 410
and other fluids according to the protocols, and controls sample
movement, pressures, and temperature. The Cell Singulation module
800 can move or rotate mechanical tissue disruptor elements
comprised of without limitation a syringe plunger, pestle, Dounce
pestle, or grinder, using a z axis stepper 2110 with a rotary motor
2120 coupled through the cap 210.
[0136] A 3D CAD representation of one embodiment of a Single-Sample
Singulator System 2000 design packaged with a `skin` is shown in
FIG. 6 and another embodiment is shown in FIGS. 7, 8, 9, and 10.
Both embodiments have a two axis mechanical motion (Z axis stepper
2110 and rotary motor 2120) integrated with fluidics based on a
syringe pump ,for example, with 1.6 .mu.L resolution with a six-way
valve (C2400MP, TriContinent) controlled by control software
725.
[0137] Referring to FIG. 6, a computer 720 with an operating
system, for example, such as Windows 10 and 85 Gbytes HD (Beelink,
AP42), can run control software 725 to control the system with
display on a 10'' touchscreen 730 (eleduino, Raspberry Pi10) or on
a tablet 750. Chassis 1010 provides the framework to mount
components and the exterior case of the system.
[0138] The embodiment of the Single-Sample Singulator System 2000
shown in FIG. 6 has a fluidic subsystem 600 with a single syringe
pump 2130 with a single six-way valve 2140 to supply liquids,
pressure, or vacuum to cartridge 200 from reagent block 415. In one
embodiment, cartridge 200 has two preprocessing chambers 440 and a
single processing chamber 460. In a preferred embodiment, magnetic
processing module 900 can apply magnetic force to cartridge 200
under software control to enable the use of paramagnetic beads,
paramagnetic surfaces, paramagnetic nanoparticles, and other
magnetic or paramagnetic particles to purify and analyze single
cells 1000, including stem and other types of cells, nuclei 1050,
microtissues 6001, organoids 6002, nucleic acids 1072, biomolecules
1070, subcellular components 1060, or other products.
[0139] A preferred embodiment of the Single-Sample Singulator
System 2000 with a case on is shown in FIG. 7. This embodiment has
a reagent module 1430 which can be separate from Single Sample
Singulator Instrument 2050 as shown in FIG. 7 with power and
control provided by Single Sample Singulator Instrument 2050 or a
separate power source and processor can be used or as shown in FIG.
6 reagent module 1430 be integrated inside a single instrument
case.
[0140] As shown in FIG. 8A, in a preferred embodiment reagent
module 1430 has reagent Peltier 1420 attached to temperature
distribution plate 1421. The temperature of reagent Peltier 1420
can be changed under control of computer 720 and control software
725 to heat or cool temperature distribution plate 1421 inside
reagent storage chamber 1419 by monitoring temperature sensor 1417,
which may be a thermocouple, or a thermistor, or optical detection
of a thermochromic surface or other method. In a preferred
embodiment, as shown in FIG. 8A, reagent Peltier 1420 maintains a
set of reagents 411 at 4.degree. C. in temperature-controlled
reagent storage chamber 1419 and room temperature reagent storage
chamber 1418 maintains a second set of reagents 411 at ambient
temperature. It will be obvious to one skilled in the art that
embodiments can have a one or more temperature controlled chambers
containing one or more reagents.
[0141] Referring to FIG. 8B, reagent storage chamber 1419 has
insulation 1422 and lid 1423. Fluidic bundle 1424 fluidically
connects syringe pump 2130 with reagent module 1430. In one
embodiment, a power and control bundle 1425 from reagent Peltier
relay board 2240 on Single Sample Singulator Instrument 2050
controls reagent Peltier 1420. In another embodiment, reagent
module 1430 is powered by separately plugging into electrical power
and reagent Peltier 1420 is controlled by a separate
microprocessor, allowing reagent module 1430 to operate
independently of Single Sample Singulator Instrument 2050 and is
connected fluidically by fluidic bundle 1424 comprised of tubing
such as 1/16 ID tygon tubing or other tubing, capillaries,
microchip, or other fluidic vessels. In some embodiments, reagent
container 1426 has reagent container lid 1427 contains one or more
reagent container sensors 1428 to monitor the amount of reagent in
the container, for example by weight, or by an phase interface
using optics or other electromagnetic measurement methods, or by
conductivity, or to determine the identity of reagent container
1426 by RFID, EEPROM, or other identification technologies.
Information from reagent container sensor(s) 1428 can be stored in
system log or be used to alert users to issues with reagent
container sensor 1428 or other actions such as the need to changes
reagents 411. In some embodiments, reagent container lid 1427 has
one or more openings that may allow tubing or capillaries or
fittings to be inserted or a hole with an optional filter. In a
preferred embodiment, reagent module 1430 has reagent Peltier
exhaust duct 1417.
[0142] Referring to FIG. 9, in a preferred embodiment, Single
Sample Singulator Instrument 2050 has z-axis stepper motor 2110,
which may have an optional encoder, that controls the vertical
position of rotary motor 2120 mounted on z-axis stepper slide 2111
attached to the inverted `U` shaped structural frame 1020 mounted
on chassis 1010. A force gauge can be incorporated into the z-stage
stepper 2110 to provide force-feedback control of the mechanical
force on the specimen 101; this can help ensure very gentle
mechanical processing steps. Syringe pump 2130 connects fluidically
with tubing or capillaries or microchips or other fluidic
connectors with six-way valve 2141 and six-way valve 2142 to supply
reagents, pressure, or vacuum to cartridge 200 (not shown) from
reagent module 1430.
[0143] Cartridge 200 is placed into cartridge receiver tray 1510 on
cartridge slide 1450 which is designed to hold cartridge 200
precisely, with the center of preprocessing chamber 440 concentric
with the center of rotary motor shaft 2121 of rotary motor 2120
within a distance or 1 or, 5, or 10, or 15, or 20, or 25, or 50, or
100, or 250 .mu.m, or more when inserted by moving cartridge 200 in
cartridge receiver tray 1510 on cartridge slide 1450 on cartridge
slide rail 1480 until spring-loaded cartridge slide knob 1452 locks
into place into a hole in cartridge slide 1450 with cartridge 200
held in place near or in contact with the thermal transfer plate
1470 and making fluidic connections with the pogo pins 1415 of
cartridge interface 1500.
[0144] The temperature regulating subsystem 1475 can set the
thermal transfer plate 1470 to a given temperature by cartridge
Peltier 1440 or other temperature regulating device such as strip
resistive heaters, circulating fluids, etc. to set the cartridge
temperature in the preprocessing chamber 440 and processing chamber
460 under control of board 2250. In some embodiments, the
temperature of the preprocessing chamber 440 and processing chamber
460 can be set independently.
[0145] In a preferred embodiment, fluidic ports on cartridge 200
dock with spring-loaded pogo pins 1415 to connect fluids, gases, or
vacuum to cartridge 200 on cartridge insertion. In another
embodiment, pogo pins 1415 or canula 1416 are moved to connect with
cartridge 200 after cartridge insertion. In another embodiment,
canula 1416 connected to fluidic lines from syringe pump 2130 are
held rigidly attached to the thermal transfer plate 1470 or other
part of instrument and cartridge 200 has flexible materials on
cartridge ports that seal with the canula(s) 1416 after cartridge
insertion, as described below. Cartridge ports are ports opening
out of a cartridge. A cartridge port may communicate directly with
a chamber by being a port in the chamber, or indirectly.e.g.,
through another chamber comprising the port and communicating with
the chamber in question.
[0146] The embodiment of the single-sample Singulator System 2000
shown in FIG. 9 has a Magnetic Processing Module 900 and magnet 910
is moved by magnetic actuator 935 mounted on inverted `U` shaped
structural frame 1020 under control of control software 725 using
controller 2122. Magnet 910 can be far from cartridge 200 as shown
in FIG. 9 and not interact with any magnetic beads 685 in cartridge
200 or in an extended position magnet 910 is moved to be near
cartridge 200 for magnetic capture and processing of magnetic beads
685.
[0147] Referring to FIG. 10, in a preferred embodiment, the
Single-Sample Singulator System 2000 has a back structural frame
1021 on structural frame 1020 that mounts electronics 710
comprising rotary motor controller 2122, z-axis stepper controller
2112, 24 V to 5 V step down power supply 2230 and 24 V to 12 V step
down power supply 2225. Power can be supplied to single-sample
Singulator System 2000 by plugging a 24 V power supply into plug
762 connecting to fuse 761 and power switch 760. Six way valves
2141 and 2142 are controlled by boards 2210 and 2212. Reagent
Peltier relay board 2240 can control reagent Peltier 1420.
[0148] Singulator systems that process one or more cartridges
simultaneously are within the scope of the present invention. FIG.
11 illustrates a Two Sample Singulator instrument 2200 that can
process two specimens 101 in two cartridges 200. The embodiment
shown in FIG. 11 has two z-axis stepper motors 2110 that
independently controls the vertical position of two rotary motors
2120 mounted on two z-axis stepper slides 2111 attached to the
inverted `M` shaped structural frame 1025 mounted on chassis 1010.
Syringe pump 2130 connects fluidically with tubing or capillaries
or microchips or other fluidic connectors with six-way valve 2141
and six-way valve 2142 to supply liquids, pressure, or vacuum to
cartridges 200 from reagent module 1430 (not shown) through pogo
pins 1415 (not shown) mounted above thermal transfer plate
preprocessing chamber 440 and processing chamber 460. A third 6 way
valve (not shown) can provide fluids to the second cartridge
interface 1500.
[0149] The cartridge 200 can have one or more Pre-Processing
Chamber(s) 440 and none, one, or more Processing Chamber(s) 460 as
well as none, one or more other chambers such as cartridge waste
chamber 435 or vacuum trap chamber 468.
[0150] In a preferred embodiment, illustrated in FIGS. 12 and 13,
cap 210, alternatively referred to as a tissue disruptor, is placed
on top of preprocessing chamber 440 after specimen 101 or tissue
specimen 120 is added through sample inlet port 425 into
preprocessing chamber 440 of cartridge 200. After cartridge 200 is
inserted into the instrument, pogo pins 1415, canula 1416, or other
fluidic connectors can connect with none, one, or more of cartridge
ports 470 to supply reagents to preprocessing chamber 440,
cartridge port 485 to supply reagents or vacuum to processing
chamber 460, and cartridge vacuum trap port 467 to supply vacuum to
vacuum trap chamber 468.
[0151] A preferred embodiment illustrated in FIG. 12 fluidically
connects preprocessing chamber 440 to processing chamber 460 using
fluidic line 453, which can be tubing, connecting from
preprocessing chamber nipple 471 to lid nipple 452 positioned over
strainer 2711 inserted into processing chamber 460, eliminating the
need for a separate strain chamber 450. In other embodiments,
strainer 2711 can be incorporated as an in-line filter, for example
in a swinney filter holder 347 attached to the output of
preprocessing chamber 440 or in fluidic line 453 or attached to lid
462. Lid 462 produces a vacuum tight seal of processing chamber 460
and vacuum trap chamber 468 when cap 465 is closed onto lid 462,
and can be attached to cartridge body 201 by ultrasonic welding,
glue, epoxy, adhesives, and other methods to produce a vacuum tight
seal and prevents changing strainer 2711 ensuring single usage of
cartridge 200.
[0152] In some embodiments, cartridge 200 can have on-cartridge
valves which can be pinch valves 491 on fluidic lines such as
fluidic line 453 which the instrument actuates to open and close
lines, or by using a `T` junction and two lines, rout fluids down
different paths such as to a optics imaging system 520. In another
embodiment, fluidic lines such as fluidic line 453 can be partially
closed to create a variable orifice 2160 that can disrupt partially
dissociated tissue. Actuators can open and pinch close tubing in
the cartridge 200, or operate the variable orifice 2160 using
variable orifice device 2150 when desired. In other embodiments,
cartridge 200 can have on-cartridge valves which can be
miniaturized pneumatic valves, or microvalves. In some embodiments,
microfluidics or microchips are used for fluidic lines. In a
preferred embodiment there are no valves on the cartridge 200 with
all fluidic control from the instrument.
[0153] Referring to FIG. 13A, when vacuum is applied to vacuum trap
port 467 or to reagent port 485, liquids including single cell
suspensions 1000, nuclei 1050, and other subcellular components
1060, and biomolecules 1070 are pulled from preprocessing chamber
440 through fluidic line 453 and strainer 2711 into strain drain
451 and into output collector region 461 of processing chamber 460.
Strainer 2711 can have pore sizes such as 2, 5, 10, 15, 20, 25, 30,
40, 50, 70, 100, 125, 200 .mu.m, or larger to filter the suspension
of biological material. Muiltiple in-line or stacked strainers 2711
can be employed to successively remove different sized components
of the dissociated tissue specimen 110. Cap 210 with cap coupler
211, and head 218 is shown ready to be inserted into sample inlet
port 425. Head 218 can have a surface for disrupting tissue that
can comprise raised features 355 that aid in mechanically
disrupting a tissue, organ, microtissue 6001, organoid 6002 or
other biological material.
[0154] Referring to FIG. 13B and FIG. 13C, the cap coupler (also
referred to as "drive head") 211 is held inside cap sheath 212
which in one embodiment has cap sheath hole 214. Cap coupler 211 is
attached to cap shaft 216 which passes through cap sheath hole 214
and is attached to the head 218 which can be a rotor 353 with
grinding teeth 355. The assembly of cap coupler 211 attached to cap
shaft 216 and head 218 are referred to as a plunger 336 which is a
type of moveable mechanical tissue disruptor 345.
[0155] Referring to FIG. 14A, in a preferred embodiment, head 218
attached to cap shaft 216 has a outwardly annular beveled head
feature 356 designed to improve centricity of head 218 inside
preprocessing chamber 440 and thereby the uniformity of side gap
221 at the bottom of travel. When z-axis stepper motor 2110 lowers
and cap coupler 211 is pushed down by rotary motor coupler 2125,
head 218 will lower until outwardly annular beveled feature 356
engages with inwardly annular beveled preprocessor chamber feature
357 on the inside wall of preprocessing chamber 440 to produce a
centered head 218 as shown in FIG. 14B. The centering of head 218
will produce a uniform side gap 221 between head 218 and the inner
wall of preprocessing chamber 440. In addition, if the height of
head 211 is less than the height of the preprocessing chamber 440
below inwardly beveled feature 357, the engagement of outwardly
annular beveled head feature 356 with inwardly annular beveled
preprocessor chamber feature 357 will set a uniform bottom gap 222.
The size of the side gap and the bottom gap can be optimized for
different cell types or for different sized nuclei or subcellular
organelles, or multicellular structures such as intestinal crypts.
In addition, to allow passage of disrupted tissue when head 218 is
seated on inwardly annular beveled preprocessor chamber feature
357, the inwardly annular beveled preprocessor chamber feature
357can be fluted to have sections with the same or different
depths. The side gap 221 between the head 218 of moveable
mechanical disruptor 345 and the inside wall is preferrably greater
than or equal to 1 .mu.m, or 2 .mu.m, or 5 .mu.m, or 10 .mu.m, or
15 .mu.m, or 20 .mu.m, or 25 .mu.m, or 30 .mu.m, or 40 .mu.m, or 50
.mu.m, or 75 .mu.m, or 100 .mu.m, or 150 .mu.m, or 200 .mu.m, or
250 .mu.m, or 500 .mu.m, and 1000 .mu.m or more, as well as any
size in between. The bottom gap 222 between the bottom of head 218
of moveable mechanical disruptor 345 and the bottom of
preprocessing chamber 440 is preferrably greater than or equal to 1
.mu.m, or 2 .mu.m, or 5 .mu.m, or 10 .mu.m, or 15 .mu.m, or 20
.mu.m, or 25 .mu.m, or 30 .mu.m, or 40 .mu.m, or 50 .mu.m, or 75
.mu.m, or 100 .mu.m, or 150 .mu.m, or 200 .mu.m, or 250 .mu.m, or
500 .mu.m, and 1000 .mu.m or more, as well as any size in between.
In some embodiments, different heads can be selected to be used
with the same diameter preprocessing chamber 440 to produce
different side gaps 221 or bottom gaps 222 to simplify
manufacturing and inventory management requirements. A bottom gap
between a flat surface of the head and the flat bottom surface of
the preprocessing chamber can also be limited by the position of
the flutes, or half domes, or other structures that prevent or
define gaps between a flat surface of the head and the flat bottom
surface of the preprocessing chamber.
[0156] Referring to FIG. 15, none, one, or more of the ports to
cartridge 200 can have flexible or low durometer port covers 442,
for example without limitation 40 to 100 durometer. As illustrated
in FIG. 15A and in cutout FIG. 15B, port cover 442 can be inserted
into the space between the port and port cover retaining cylinder
441 to secure the port cover 442 in place over, for example as
shown, reagent addition port 470. A fluidic canula 1416 or fluidic
pogo pin 1415 with an outside diameter larger than port cover
center hole 446 can engage the port covered by port cover 442 and,
because of the relatively low durometer, the port cover 442 will be
deformed by fluidic canula 1416 or fluidic pogo pin 1415 to create
a seal around the fluidic canula 1416 or fluidic pogo pin 1415. In
some configurations, the deformation can be used to eliminate the
need for springs and the use of the fluidic pogo pin 1415 can be
replaced by a non-movable fluidic canula 1416. FIG. 15C shows port
cover 442 retained by crimp seal 443. FIG. 15D shows port cover 442
retained by forming port cover retaining cylinder 442 higher than
the port cover 442 and melting the port cover retaining cylinder
442 to form a heat stake lip 444 that retains port cover 442.
[0157] In a preferred embodiment the Single Sample Singulator
Instrument 2050 has an actuator for mechanical processing that has
a stepper motor 2110 that controls the vertical position of rotary
motor 2120 and rotary motor shaft 2121 attached to rotary motor
coupler 2125 that in turn can mechanically couples with cap coupler
211 of the cap 210 when inserted into cartridge 200. The coupler
can have a drive head that takes any appropriate form, such as a
slot, a phillips head, a quadrex, atri-wing, aspanner or a hex.
Rotary motor coupler 2125 has one or more facets that reversibly
engage cap coupler 211 by actions such as moving downward and
slowly rotating. As shown in FIG. 16 A, in a preferred embodiment,
rotary motor coupler 2125 has a single blade to engage cap coupler
211 in cap 210. As shown in the cutaway in FIG. 16 B, when stepper
motor 2110 lowers, the rotary motor coupler 2125 attached to rotary
motor shaft 2121 engages cap coupler 211 in cap 210 and if the
rotary motor coupler 2125 is not lined up with cap coupler groove
217, the rotary motor coupler 2125 can not directly insert into the
cap coupler groove 217. In a preferred embodiment, cap coupler 211
has two surfaces on either side of cap coupler groove 217 which
slope in opposite directions across the cap coupler 211 such that
each side has a higher and lower wall on either side of cap coupler
groove 217. When rotary motor shaft 2121 turns in the clockside
direction (looking from above), rotary motor coupler 2125 blade
spins in the clockside direction and encounters the high side of
the wall of cap coupler groove 217 and begin to rotate cap coupler
211 clockwise. As stepper motor 2110 lowers, the rotary motor
coupler 2125 will engage the cap coupler groove 217, as shown in
FIG. 16C. As shown in FIG. 16D,when stepper motor 2110 continues to
lowers, the rotary motor 2120 and rotary motor shaft 2121 attached
to rotary motor coupler 2125 will lower, pushing on cap coupler
groove 217 and the cap coupler 211 will compress cap spring 213
against the bottom of cap sheath 212 and lower head 218. As shown
in FIG. 16E, head 218 can be lowered close to or in contact with
the bottom of preprocessing chamber 440, which can be a stator 354,
and head 218 can be rotated to disrupt tissue. When stepper motor
2110 raises, rotary motor 2120 and rotary motor coupler 2125 raise
up and cap spring 213 decompresses to push cap coupler 211 against
rotary motor coupler 2125 to continue engagement.
[0158] In another embodiment of the Single Sample Singulator
Instrument 2050, stepper motor 2110 controls the vertical position
of rotary motor 2120 which is magnetically coupled to moveable
disruptor 345 with a magnetic or paramagnetic element embedded with
cap 210 as part of cap coupler 211 or as part of moveable disruptor
345 or head 218.
[0159] When rotary motor coupler 2125 is engaged with cap coupler
211 by mechanical coupling, magnetic coupling, pneumatic, or
fluidic coupling, or other coupling methods, and rotary motor 2120
rotates, moveable disruptor 325 and head 218 are rotated. Stepper
motor 2110 controls the vertical position of the rotary motor 2120
and thereby the the vertical position of rotary motor coupler 2125,
to raise or lower moveable disruptor 345 and head 218 in
preprocessing chamber 440. Combining rotation of rotary motor 2120
and movement of stepper motor 2110 enables many patterns of motion
of moveable tissue disruptor 345 and head 218.
[0160] The inside walls of preprocessing chamber 440 can be
embodied in many different shapes. The inside walls of
preprocessing chamber 440 can be fluted to have sections with
different depths. In a preferred embodiment, the inside wall can
have a circular profile with the largest gap between the head 218
of moveable mechanical tissue disruptor 345 and the inside wall of
preferrably greater than or equal to 1 .mu.m, or 2 .mu.m, or 5
.mu.m, or 10 .mu.m, or 15 .mu.m, or 20 .mu.m, or 25 .mu.m, or 30
.mu.m, or 40 .mu.m, or 50 .mu.m, or 75 .mu.m, or 100 .mu.m, or 150
.mu.m, or 200 .mu.m, or 250 .mu.m, or 500 .mu.m, and 1000 .mu.m or
more, as well as any size in between.
[0161] Moveable tissue disruptor 345 can be embodied in many
different shapes with many different profiles. In one embodiment,
moveable tissue disruptor 345 can have a head 218 which is a rotor
353 with optional features, for example, grinding teeth 355 on the
bottom of rotor 353 and grinding teeth 355 on stator 354 which is
on the top surface of the bottom of the preprocessing chamber 440
to assist in disruption of large pieces of tissue specimens 120
into smaller pieces or assist in the dissociation into single cells
1000 or nuclei 1050 or biomolecules 1070. As shown in FIGS. 13 and
14, the sides of head 218 can be a cylinder to create an inside gap
221 with the inside wall over the length of the cylinder. By
raising and lowering head 218 without turning head 218, thereby
using it as a moveable disruptor 345, the system can process
specimen 101 by trituration. In another embodiment the sides of the
head 218 can form a ball-like structure to create a gap with the
inside wall in a small area and the bottom of preprocessing chamber
440 can be rounded to match the ball-like structure to create a
Dounce-like mechanical tissue disruptor 345. In other embodiments,
multiple regions with gaps of the same or different sizes can be
created by varying the side profile of moveable tissue disruptor
345 and the inner wall of preprocessing chamber 440.
[0162] Disruption of tissue can include a plurality of disruption
steps, each involving positioning the head a different distance
from floor of the chamber to produce gaps of different sizes.
Typically, at each position, the head will rotate, further
facilitating disruption or mixing. In certain embodiments, an organ
can be auto-minced by the disrutor before tissue disruption into
single cells 1000 or nuclei 1050 or other biological materials.
Such a method can involve a first disruption step, which can
include setting the head at a plurality of different distances from
the floor of the chamber and rotating at each gap distance, to
provide tissue with greater surface area and less distance for
access by enzymes. A next step can involve incubating the
auto-minced organ with enzymes or chemicals for tissue disruption
into single cells 1000 or nuclei 1050. A next step can involve a
second disruption step, which, in turn, can include setting the
head at a plurality of different distances from the floor of the
chamber and rotating the head.
[0163] Example: Production of a Single Cell Suspension from Fresh
Mouse Kidney.
[0164] The Single Sample Singulator System 2000 can be operated in
many configurations. As an example, an operator may wish to process
a fresh mouse kidney specimen 101 into a single cell suspension
1000 and use reagents stored on Reagent Module 1430. The operator
would remove cap 210 from cartridge 200 as shown in FIG. 13A and
add a whole mouse kidney, or a part of mouse kidney, or part of a
kidney that had been preminced to sample inlet port 425. The cap
210, which is a moveable disruptor, is replaced on preprocessing
chamber 440 with the bottom of cap sheath 212 seated on an annular
seat in preprocessing chamber 440. The now complete cartridge with
a tissue specimen is placed on cartridge receiver tray 1510 and
inserted into the Single Sample Singulator instrument 2050 with
cartridge slide 1450. After the appropriate protocol is selected
through user interface 740 on tablet 750, the Single Sample
Singulator instrument 2050 heats thermal transfer plate to hold the
preprocessing chamber 440 at 37.degree. C. and then begins
processing kidney specimen 101.
[0165] After initialization of electronic boards, the z-axis
stepper motor 2110 moves the rotary motor 2120 down to engage
rotary motor coupler 2125 with cap coupler 211.
[0166] The control software 725 then selects the proper valve
settings to pull two mL of mouse kidney reagent solution from
Position 3 in temperature-controlled reagent storage chamber 1419
of reagent module 1430, as shown in FIG. 17, and deliver it through
port 470 to preprocessing chamber 440 where the mouse kidney has
been placed.
[0167] If selected by the protocol, an auto-mince procedure to
macerate the tissue is performed by the z-axis stepper motor 2110
moving rotary motor 2120, and therefore the mechanical tissue
disruptor and head 218, which is functioning as a rotor 353, to 1.5
mm from the bottom of the preprocessing chamber 440 and then rotor
353 is rotated clockwise for four seconds and then counterclockwise
for four seconds at 95 rpm. Rotor 353 is lowered to 0.6 mm from the
bottom and rotated clockwise for four seconds and counterclockwise
for four seconds at 95 rpm. Rotor 353 is lowered to 0.3 mm from the
bottom and rotated clockwise for four seconds and counterclockwise
for four seconds at 95 rpm to complete the standard automince
portion of the protocol.
[0168] For mouse kidney, the now auto-minced kidney specimen 101 is
then incubated for twenty minutes with continuous top immersion
mixing where rotor 353 is lowered into the top third of the mouse
kidney reagent solution with kidney specimen 101 in preprocessor
chamber 440 and the rotary motor 210 spins rotor 353 clockwise at
95 rpm in a continuous immersion mixing mode while the enzymatic
formulation digests the extracellular matrix in the solid tissue to
release cells.
[0169] After 20 min, the tissue is mechanically disrupted by
lowering rotor 353 until it is 4.2 mm from the bottom,
approximately 20% immersed into mouse kidney reagent solution with
kidney specimen 101, and then the first mechanical disruption cycle
is performed with rotor 353 rotating clockwise for four seconds and
then rotating counterclockwise for four seconds at 95 rpm. The
second disruption cycle is performed by lowering rotor 353 by 1.5
mm and rotating clockwise for four seconds and then
counterclockwise for four seconds at 95 rpm. The third disruption
cycle is by lowering rotor 353 by 0.9 mm and and rotating clockwise
for four seconds and counterclockwise for four seconds at 95 rpm.
Then, the fourth and fifth disruptions cycles are performed with
lowering rotor 353 by 0.6 mm each cycle with rotation clockwise for
four seconds, counterclockwise for four seconds, then rotation
clockwise for four seconds, and counterclockwise for four seconds
at 95 rpm for each disruption cycle. For the sixth disruption
cycle, the rotor 353 is raised 0.3 mm and then rotated clockwise
for four seconds, counterclockwise for four seconds, clockwise for
four seconds, and counterclockwise for four seconds at 95 rpm. For
the seventh disruption cycle, the rotor 353 is lowered 0.6 mm and
rotated clockwise for four seconds, counterclockwise for four
seconds, rotated clockwise for four seconds, and counterclockwise
for four seconds at 95 rpm. For the eight and final disruption
cycle, the rotor 353 is lowered 0.3 mm in contact with the bottom
surface of preprocessing chamber 440 and rotated clockwise for four
seconds, counterclockwise for four seconds, rotated clockwise for
four seconds, and counterclockwise for four seconds at 95 rpm. Many
other possible disruption profiles are enabled by this instant
invention.
[0170] The mechanical tissue disruption occurs at two places:
first, at the bottom of rotor 353 by grinding teeth 355 and the top
of stator 354 with complementary grinding teeth 355 to mechanically
dissociate the solid tissue in bottom gap 222 and secondly, the gap
between the circumference of the rotor 353 and the inner wall of
preprocessing chamber 440 acts as an orifice to disrupt the
tissue.
[0171] With the rotor 353 positioned at the bottom of preprocessing
chamber 440, syringe pump 2130 then pulls vacuum through the
appropriate six way valve settings on vacuum trap port 467 to pull
the dissociated mouse kidney single cell suspension through line
453, through 70 .mu.m strainer 2711 where it drains down strain
drain 451 and into output collector region 461 in processing
chamber 460.
[0172] The control software 725 sets the selection of valve
settings to pull two mL of HBSS-Ca-Mg from Position 13 in room
temperature reagent storage chamber 1418 of reagent module 1430 as
shown in FIG. 17 and deliver it through port 470 to preprocessing
chamber 440. Rotor 353 can be moved to mix any remaining
dissociated cells with the HBSS-Ca-Mg and then with rotor 353
positioned at the bottom of preprocessing chamber 440, syringe pump
2130 then pulls vacuum through the appropriate six way valve
settings on vacuum trap port 467 to pull the HBSS-Ca-Mg and any
remaining dissociated mouse kidney single cells suspension through
line 453, through 70 .mu.m strainer 2711, down strain drain 451 and
into output collector region 461 in processing chamber 460. This
process is then repeated to deliver and pull a second two mL of
HBSS-Ca-Mg through preprocessing chamber 440 and into processing
chamber 460. The mouse kidney single cell 1000 suspension can then
be pipetted out by opening processing chamber cap 465 and
withdrawing the cell suspension from output collector region 461
using a pipettor.
[0173] The mouse kidney single cell 1000 suspension can be
centrifuged at 300g for five min to collect the cells as a pellet,
the red blood cells lyzed for five min with a RBC lysis buffer, and
the suspension centrifuged at 300 g for five min to collect the
cells. As an example, a 262 mg of mouse kidney produced a single
cell suspension 1000 by this process with a cell titer of
14,670,000 cells at a 85.5% viability as determined by counting on
a Countess II with Trypan blue staining as shown in FIG. 18A.
[0174] Other tissues or organs may benefit from different modes of
mixing. The Single Sample Singulator System 2000 is designed to
perform a plurality of mixing modalities. For example, top mixing
is designed to position the bottom of head 218 at 15 mm above the
bottom and rotate head 218 to mix the enzymatic or chemical
dissolution solution 410 with the specimen 101. Shallow immersive
mixing can be performed by continuously rotating head 218 as it is
moved from 17.7 mm above the bottom down to 16.8 mm and back up
again. Tritutation mixing can be performed by moving head 218
without rotation from 12.3 mm above the bottom down to 0.3 mm above
the bottom. Many other mixing modalities are enabled.
[0175] Example: Production a Single Nuclei Suspension from Flash
Frozen Human Brain.
[0176] The Single Sample Singulator System 2000 can be operated in
many configurations to produce nuclei 1050 suspensions. As an
example, an operator may wish to process a fresh mouse kidney
specimen 101 into a single nuclei suspension 1050 and use reagents
stored on Reagent Module 1430. The operator would remove cap 210
from cartridge 200 as shown in FIG. 13 and add a whole mouse
kidney, or a part of a kidney, or part of a kidney that had been
preminced to sample inlet port 425. The cap 210, which is a tissue
disruptor, is replaced on preprocessing chamber 440 and the now
complete cartridge with a tissue specimen 101 is placed on
cartridge receiver tray 1510 and inserted into the Single Sample
Singulator instrument 2050 with cartridge slide 1450. After the
appropriate protocol is selected through user interface 740 on
tablet 750, the Single Sample Singulator instrument 2050 cools
thermal transfer plate 1470 to hold the preprocessing chamber 440
and processing chamber 460 at 4.degree. C. and then begins
processing kidney specimen 101. The thermal transfer plate 1470 can
also be preheated or precooled as needed.
[0177] After initialization of boards, the z-axis stepper motor
2110 moves the rotary motor 210 down to engage rotary motor coupler
2125 with cap coupler 211. The control software 725 then selects
the valve settings to pull two mL of nuclei isolation solution 412
from Position 1 in temperature-controlled reagent storage chamber
1419 of reagent module 1430 as shown in FIG. 17 and deliver it
through port 470 to preprocessing chamber 440.
[0178] The tissue is then mechanically disrupted by lowering head
218 which will function as rotor 353 until it is 4.2 mm from the
bottom, approximately 20% immersed into the nuclei isolation
solution 412 with kidney specimen 101, and then the first
mechanical disruption cycle is performed with moveable mechanical
disruptor 345 and head 218 acting as a rotor 353 rotated clockwise
for four seconds and then rotated counterclockwise for four seconds
at 135 rpm. The second disruption cycle is by lowering rotor 353 by
1.5 mm and rotating clockwise for four seconds and then
counterclockwise for four seconds at 135 rpm. The third disruption
cycle is by lowering rotor 353 by 0.9 mm and and rotating clockwise
for four seconds and then rotating counterclockwise for four
seconds at 135 rpm. Then, the fourth and fifth disruptions cycles
are performed with lowering rotor 353 by 0.6 mm with rotation
clockwise for four seconds, counterclockwise for four seconds,
rotation clockwise for four seconds, and counterclockwise for four
seconds at 135 rpm for each disruption cycle. For the sixth
disruption cycle, the rotor 353 is raised 0.3 mm and then rotated
clockwise for four seconds, counterclockwise for four seconds,
clockwise for four seconds, and counterclockwise for four seconds
at 135 rpm. For the seventh disruption cycle, the rotor 353 is
lowered 0.6 mm and rotated clockwise for four seconds,
counterclockwise for four seconds, rotated clockwise for four
seconds, and counterclockwise for four seconds at 135 rpm. For the
eigth disruption cycle, the rotor 353 is lowered 0.3 mm and rotated
clockwise for four seconds, counterclockwise for four seconds,
rotated clockwise for four seconds, and counterclockwise for four
seconds at 135 rpm.
[0179] The mechanical tissue disruption again occurs both at the
bottom of rotor 353 by grinding teeth 355 and the top of stator 354
with complementary grinding teeth 355 mechanically dissociating the
solid tissue in bottom gap 222 as well as any tissue passing
between the circumference of the rotor 353 and the inner wall of
preprocessing chamber 440 in side gap 221.
[0180] With the rotor 353 positioned at the bottom of preprocessing
chamber 440, syringe pump 2130 then pulls vacuum through the
appropriate six way valve settings on vacuum trap port 467 to pull
the dissociated mouse kidney nuclei suspension through line 453,
through a 40 .mu.m strainer 2711 in processing chamber 460, down
strain drain 451 and into output collector region 461.
[0181] The control software 725 sets the selection of valve
settings to pull two mL of nuclei storage solution 413 from
Position 2 in temperature-controlled reagent storage chamber 1419
of reagent module 1430 as shown in FIG. 17 and delivers it through
port 470 to preprocessing chamber 440. Rotor 353 can be moved to
mix any remaining dissociated nuclei 1050 with the nuclei storage
solution 413 and then with rotor 353 positioned at the bottom of
preprocessing chamber 440, syringe pump 2130 pulls vacuum through
the appropriate six way valve settings on vacuum trap port 467 to
pull the nuclei storage solution 413 and any remaining dissociated
mouse kidney single nuclei 1050 suspension through line 453,
through a 40 .mu.m strainer 2711, down strain drain 451 and into
output collector region 461. The mouse kidney single nuclei 1050
suspension can then be pipetted out by opening processing chamber
cap 465 and withdrawing the cell suspension from output collector
region 461.
[0182] The mouse kidney single cell 1050 suspension can be
centrifuged at 500 g for 5 min to collect the cells as a pellet
before resuspension in nuclei storage solution 413 or other media.
As an example, a 108 mg mouse kidney produced by this process
yielded a nuclei suspension 1050 with a titer of 24,225,000 as
determined by counting on a Countess II with Trypan blue staining;
a picture of the nuclei suspension 1050 is shown in FIG. 18B.
[0183] Example: Processing FFPE Tissue into Cells or Nuclei
[0184] FFPE tissue is commonly used by pathologists to examine
biopsy samples. Massive banks of FFPE tissue contain archives of
tissue samples from many disease states including cancers.
Currently, isolating single cells or nuclei from FFPE is
challenging and not automated.
[0185] In one embodiment, one or more thin sections from an FFPE
block are added into cartridge 200, the cap 210 added, and the
cartridge 200 placed into the Single Sample Singulator instrument
2050. In some embodiments cartridge 200 has a filter, such as a 25
.mu.m filter added in or over the channel leading to preprocessing
chamber nipple 471 to prevent loss of the undissociated FFPE thin
section through the preprocessing chamber nipple 471.
[0186] After selection of the appropriate cell or nuclei FFPE
protocol, and using the appropriate setup of reagent module 1430,
the instrument can add, for example, 2 mL of xylol from the reagent
module 1430 and incubate for a time period selected from the range
of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min or longer
at room temperature or other temperature. The xylol is then pulled
into the processing chamber 460 as described and the process
repeated two additional times with xylol. Xylene, histolene, and
other compatible solvents can replace xylol. In some embodiments,
the volume of processing chamber 460 is enlarged to accommodate the
deparafinnization materials. In other embodiments, a separate waste
chamber is added and pinch valves 491 are used to direct flow
either to a waste chamber or processing chamber 460.
[0187] The instrument can then perform reverse sequential ethanol
washes, for example, by adding two mL of 100% ethanol from the
reagent module 1430 to cartridge 200 and incubating for a time
period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10
min, 15 min, 30 min or longer at room temperature or other
temperature. The 100% ethanol is then pulled into the processing
chamber 460 as described and the process repeated none, one, or
more additional times with 100% ethanol.
[0188] The instrument can add 2 mL of 70% ethanol from the reagent
module 1430 to cartridge 200 and incubate for a time period
selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15
min, 30 min or longer at room temperature or other temperature. The
70% ethanol is then pulled into the processing chamber 460 as
described and the process repeated none, one, or more additional
times with 70% ethanol.
[0189] The instrument can add 2 mL of 50% ethanol from the reagent
module 1430 to cartridge 200 and incubate for a time period
selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15
min, 30 min or longer at room temperature or other temperature. The
50% ethanol is then pulled into the processing chamber 460 as
described and the process repeated none, one, or more additional
times with 50% ethanol. In some embodiments, a 30% ethanol step or
other additional reverse sequential ethanol wash steps can be
added. In some embodiments, the ethanol washes and other solutions
can be supplemented with PBS, bovine serum albumin, RNAse
inhibitors, protease inhibitors, or other supplements.
[0190] The instrument can add 2 mL of purified water, such as
double distilled water with RNAse inhibitors, from the reagent
module to cartridge 200 and incubated for a time period selected
from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30
min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at at
4.degree. C., room temperature or other temperatures. The water is
then pulled into the processing chamber 460 as described and the
process repeated none, one, or more additional times with purified
water. The waste in processing chamber 460 can be removed at this
time or previously as needed if it has not been directed to a waste
chamber.
[0191] The deparaffinized FFPE can then be treated by different
methods. In one method, an enzymatic digestion is performed by
adding up to two mL of proteinase K solution (0.005% proteinase K,
30 U/mg protein, in 50 mM Tris hydroxymethyl aminomethane
hydrochloride (pH 7.0), 10 mM EDTA, and 10 mM sodium chloride),
with optional DNase addition, and incubating for a time period
selected from the range of 1 min, 5 min, 10 min, 15 min, 30 min, 60
min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at 37.degree. C.
or up to 60.degree. C. or other temperatures.
[0192] If cells are to be produced, the proteinase K solution can
be diluted by the addition of up to 2 mL of a solution to dissolve
residual extracellular matrix such as adding formulations of a
reagents or mixture of components comprised of but not limited to
collagenases (e.g., collagenases type I, II, Ill, IV, and others),
elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutral
protease, clostripain, caseinase, neutral protease (Dispase.RTM.),
DNAse, protease XIV, RNase inhibitors, or other enzymes,
biochemicals, or chemicals such as EDTA, protease inhibitors,
buffers, acids, or base. In one embodiment, two mL of an enzymatic
cocktail containing 1 mg/ml of Collagenase/Dispase (Roche) and 100
units/ml of Hyaluronidase (Calbiochem) in PBS/0.5 mM CaCl2 are
added with optional DNase addition and incubated for a time period
selected from the range of 1 min, 5 min, 10 min, 15 min, 30 min, 60
min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at 37.degree. C.
or other temperatures. The released single cell 1000 suspension is
then pulled into the processing chamber 460 through a 70 .mu.m
filter as described and removed. The released cells are then
centrifuged at 300 rpm for 5 minutes, and resuspended in buffer,
such as PBS or HBSS, and optionally again filtered through a 70
.mu.m or other filter. Additional processing can then be performed
as appropriate for downstream procedures.
[0193] If nuclei are to be produced, 2 mL of nuclei isolation
buffer 412, such as NST buffer (146 mM NaCl, 10 mM Tris base at pH
7.8, 1 mM CaCl2, 21 mM MgCl2, 0.05% BSA, 0.2% Nonidet P-40) can be
be added to the proteinase K solution and incubated fora time
period selected from the range of 1 min, 5 min, 10 min, 15 min, 30
min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours. The
released nuclei 1050 suspension is then pulled into the processing
chamber 460 through a 40 .mu.m or other filter as described and
removed. The released nuclei are then centrifuged at 500 rpm for 5
minutes, and resuspended in nuclei storage buffer 413, and
optionally again filtered through a 40 .mu.m or other filter.
Additional processing can then be performed as appropriate for
downstream procedures.
[0194] Example Using a Vertical Cartridge in the Singulator System
to Generate Microtissues or Organoids
[0195] Another preferred embodiment of cartridge 200 is shown in
FIGS. 19 A and B. This vertical cartridge 200 is designed to be
injection molded and then sealed with a material such as a heat
sealable plastic laminate, or laser welded, or ultrasonically
welded or other means to seal cartridge 200. It has two processing
chambers 460 for processing samples which facilitates improved
mixing during processing steps.
[0196] Referring to FIGS. 19 A and B, a typical process flow is as
follows. The operator inserts tissue specimen 120 into the
preprocessing chamber 440 through sample inlet port 425 and places
cap 210 (not shown) onto cartridge 200 and inserts cartridge 200
into the Singulator System 100, Tissue Processing System 80, or
Sample Processing System 50 as described above. After selection of
the appropriate program, the instrument makes the mechanical
connection to cap 210 through rotary motor coupler 2125 (not shown)
and fluid/gas connections to the fluid/gas inlets/outlets 480. The
instrument also contacts the preprocessing chamber 440 and the two
processing chambers 461 and 462 from the back of cartridge 200 with
elements such as cartridge Peltier 1440 which can heat or cool said
fluid chambers.
[0197] The appropriate enyzmatic or chemical dissolution solution
410 is moved by fluidic subsystem 600 from reagent module 1430 into
the preprocessing chamber 440 from fluid/gas inlets/outlets 480
through fluid channel 441. The solution may be heated or cooled by
the action of the temperature regulating subsystgem 1475 engaged
with preprocessing chamber 440. The enyzmatic or chemical
dissolution solution 410 can contain enzymes or chemicals to help
dissociate the tissue specimen 120 into a cell suspension 1000 or
nuclei suspension 1050. The tissue disruptor in cap 410 acting as
grinder rotor 420 is then mechanically rotated and brought up/down
by the Singulator System 100 whereby tissue specimen 120 is
separated into smaller and smaller pieces by the action of the
grinding features on the head 218 which can be a grinder rotor 420
and grinder stator 421 on the bottom of preprocessing chamber 440.
Single cell 1000 or nuclei suspension 1050 production is achieved
by the combined action of the grinding elements and
incubation/exposure of the tissue specimen 120 to reagents 411,
e.g., enzymes, or chemicals , or combinations of enzymes and
chemicals as described herein. After the tissue disruption is
sufficiently advanced, the grinder rotor 420 can be brought
completely down until it touches the grinder stator 421 whereby the
singulated cells 1000 in the enzymatic dissolution solution 410 or
nuclei suspension 1050 in chemical dissolution solution 414 are
pushed around and above the grinder rotor 420 through the side gap
211 between the rotor and the wall of preprocessing chamber
440.
[0198] All the the fluid/gas inlets/outlets 480 are then sealed by
the syringe pump 2130 and appropriate valves and the singulated
cells 1000 or nuclei 1050 suspension, or nucleic acids 1072 are
pulled from the preprocessing chamber 440 through channel 442 to
strain chamber 450 and then through channel 443 towards processing
chamber 461 by applying negative pressure through channels 446 or
444. A filter in strain chamber 450 prevents undissociated tissue,
cell aggregates, and large debris from entering processing chamber
461. Waste chamber 431 can containing a liquid absorbent or solid
absorbent to prevent any liquid from exiting through the fluid/gas
inlets/outlets 480 and into the Singulator System 100.
[0199] To generate microtissues 6001 or organoids 6002 from a
single cell 1000 suspension, as shown in the closeup of FIG. 19 in
FIG. 20, a nozzle 6100 at the end of channel 443 can be used to
create hanging droplets 6200 which can generate microtissues 6001
or organoids 6002. This is achieved by control of the flow from
strain chamber 450 to gently pull the single cell 1000 suspension
until a droplet is formed on nozzle 6100. Processing chamber 461
can be prefilled with water or buffer with in some embodiments
saturated with 5% CO.sub.2 to provide hydration to hanging droplet
6200 to control evaporation. The temperature control of cartridge
200 by the instrument can incubate the microtissues 6001 or
organoids 6002 at the desired temperature, for example, 37.degree.
C. The incubation can be for minutes, hours or days before
harvesting of the microtissues 6001 or organoids 6002 by removing
the seal on processing chamber 461, or cutting it open depending on
cartridge design. In other embodiments, once the hanging droplet
6200 has formed microtissues 6001 or organoids 6002, the hanging
droplet 6200 in processing chamber 461 can be released by pulling
vacuum or pushing fluids through channel 442 and into channel 441.
Once the microtissues 6001 or organoids 6002 are in processing
chamber 461 the microtissues 6001 or organoids 6002 can be further
grown by suppling sterile growth media through channel 444 or other
channels.
[0200] If desired, the microtissues 6001 or organoids 6002 can be
mixed with any added reagents by applying alternative negative (and
or positive) pressure to channels 444 and 445 to move the sample
back and forth from processing chamber 461 to processing chamber
462 through channel 448. If no further processing is desired, the
operator can pull out the microtissues 6001 or organoids 6002
through an opening or processing chamber cap 465 (not shown) in the
top wall of processing chamber 461 or processing chamber 462 or by
removing the seal on the surface of the cartridge 200.
[0201] The microtissues 6001 or organoids 6002 can also be
processed in vertical cartridge 200 by use of magnetic beads for
the positive selection or depletion of specific cell types, such as
stem cells, or for washing the cells and/or for exchanging the
buffer. The microtissues 6001 or organoids 6002 can be further
processed by using cell-specific affinity reagents coupled to
magnetic beads. For example, cell-type specific affinity magnetic
beads and reaction solutions are injected through channel 444 into
processing chamber 461. The beads are incubated with the
microtissues 6001 or organoids 6002 by mixing though channel 448 as
described above, whereby the magnetic beads bind to their target
cells. Then, magnet(s) 910 is/are applied to the frontside of
processing chambers 462, whereby the magnetic beads (and attached
microtissues 6001 or organoids 6002) are attracted to and held at
the processing chamber 462 wall. The microtissues 6001 or organoids
6002 that does not contain the specific targets is pulled into the
waste chamber 432 by applying negative pressure to through channels
447 and 449 . Waste chamber 432 which can optionally contain a
liquid or solid absorbent substance.
[0202] Simultaneously or subsequently, washing solution can be
injected through channel 444 and pulled into chamber 462 by
applying vaccum on channel 446 to wash the beads attached to magnet
910 by combinations of mixing, magnetic release/application and
pulling liquid to the waste chamber 432 as described. This process
can be repeated one or more times.
[0203] After the microtissues 6001 or organoids 6002 are in the
desired output media, the magnet 910 is released, the cells
homogeneously resuspended by mixing by moving the cells back and
forth through channel 448, and then the microtissues 6001 or
organoids 6002 is pulled either into processing chamber 461 or 462.
The operator can then pull out the microtissues 6001 or organoids
6002 through an opening in the top wall of Processing Chamber 461
or 462 covered by a foil-seal, or septum, or processing chamber cap
465 or other mechanism (not shown). Other
processing/reaction/fluidic elements can be added to the cartridge
as desired to enable additional processing modes in including
without limitation tangential flow filtration, optical
interrogation, library preparation, and nucleic acid
purification.
[0204] Similar processing methods can also be used to resuspend the
microtissues 6001 or organoids 6002 in a specific media, buffer, or
growth solution, such as Matricel, or to perform labeling with
chemicals such as mass tags, or fluorescent dyes, or Raman labels,
or other labels. In addition, similar methods can be used to
chemically or biochemically modify single cells 1000 or nuclei 1050
or microtissues 6001 or organoids 6002 including screening of
potential therapeutic compounds, or inhibitors of growth or
viability. In some embodiments, Measurement Subsystem 500 can
interrogate the microtissues 6001 or organoids 6002 inside
cartridge 200.
[0205] In another embodiment, a single cell 1000 suspension is
pulled directly into processing chamber 461 and stem cells purified
by magnetic bead processing as described with non-stem cells
removed. In another embodiment, a single cell 1000 suspension is
pulled directly into processing chamber 461 and chemically induced
into stem cells, or with transcription factors, or by by
retroviral-mediated expression of the four transcription factors
Oct4, Sox2, cMyc, Klf4. Many other modalities are possible.
[0206] Example: Decreasing the Degradation of Biomolecules in
Nuclei and Subcellular Components
[0207] The degradation of RNA in nuclei during and after nuclei
isolation can alter the amount and representation of RNA. The
degradation is tissue specific and currently can prevent single
nuclei sequencing of the transcriptome from tissues with high RNAse
activity such as pancreas. Similarly, RNA or other biomolecules
from other subcellular components such as nuclei and mitochondria
can be degraded during isolation procedures. A method to improve
the quality of RNA and other biomolecules comprised of proteins,
lipids, polysaccharides, etc. isolated from solid tissue samples is
described.
[0208] Current methods to dissociate solid tissues into nuclei,
using reagents alone or in combination with mechanical disruption
techniques, can result in RNA becoming severely degraded and
therefore not useful for downstream genomic analyses. Current
methods to preserve RNA quality include the use of high
concentrations of RNAse inhibitors, performing operations at low
temperature, and performing operations quickly. The action of
RNAses on RNA within a nucleus are rapid enzymatic reactions.
Addition of RNAse inhibitors that bind to RNAses can be ineffective
for tissue types that exhibit high levels of RNAse activity.
Performing operations at 4.degree. C. can lower the rate of
enzymatic activity, but again, if there are high levels of RNAses
in the tissue sample, simply lowering the temperature, even in the
presence of RNAse inhibitors, can fail to adequately protect RNA
from degradation. Isolating nuclei from solid tissue samples in the
1-1000 mg range may also require total reagent volumes of 0.5 to 5
ml, and including RNAse inhibitor reagents at the typical one
unit/microliter concentration can cost hundreds of dollars per
sample.
[0209] This instant invention describes the use of additives to
reduce the rate of degradation. In one method, proteinase K, a
serine protease, or other proteases are added to degrade RNAses or
DNases released from the extracellular matrix or upon lysis of cell
membranes.
[0210] In another embodiment, reagents to increase the viscosity
are added during the isolation of nuclei or other subcellular
components, thereby reducing the rate of diffusion of DNases,
RNAses, lipases, nucleases, proteases, and other degradatory
enzymes, and therefore reducing the level of RNA degradation or
other biomolecule degradation during the isolation procedure for
nuclei, mitochondria or other subcellular components. Examples of
such additives include, but aren't limited to, crowding agents, and
biocompatible high molecular weight polymers comprised of ficoll,
dextran, sucrose, trehalose, cellulose, and polyethylene glycol.
Typical concentrations of such reagents used are approximately but
not limited to 0.01% to 50% w/v.
[0211] A preferred embodiment of the method applied to isolating
nuclei from solid tissues or previously prepared single cell
suspensions is to include one or more of the additives in either a
nuclei isolation solution 412, nuclei storage solution 413, or both
as used to isolate nuclei from tissue samples. For example, when
using the Singulator System 100 for isolating nuclei, the nuclei
isolation solution 412 might have 5% w/v ficoll added or the nuclei
storage solution 413 might have 5% w/v ficoll. One or both of these
solutions might also contain one or more protease inhibitors, and
one or more RNAse inhibitor reagents including but not limited to
SUPERase.cndot. In RNase Inhibitor, RNaseOUT Recombinant
Ribonuclease Inhibitor, RNAsecure RNase Inactivation Reagent,
Recombinant RNase Inhibitor and small molecule reagents including,
but not limited to nucleotides and inorganic phosphates.
[0212] A protocol for improved isolation of mouse kidney nuclei
from 300 mg of fresh or flash frozen mouse kidney tissue might be
comprised of:
[0213] 1) Loading the nuclei isolation solution 412 and nuclei
storage solution 413 with additives to increase viscosity onto the
reagent module 1430.
[0214] 2) Placing a fresh or flash frozen mouse kidney tissue
specimen 120 in a cartridge 200 precooled at 4.degree. C. and
adding tissue disruptor cap 210.
[0215] 3) Placing cartridge 200 in a Single Sample Singulator
instrument 2050 set to 4.degree. C. operating temperature.
[0216] 4) Selecting the nuclei isolation protocol from the software
user interface 740 and selecting "Run". The Singulator then
delivers 2 mL of the nuclei isolation solution 412 to the mouse
kidney tissue specimen 120 in the preprocessing chamber 440;
mechanically disrupts the tissue at 135 rpm; pulls the sample
through a 40 micron strainer into the processing chamber 460; adds
2 mL of the nuclei storage soluion 413 to preprocessing chamber 440
to rinse residual material and decrease the final detergent
concentration to quench disruption; pulls the added nuclei storage
solution 413 through the filter into the processing chamber
460.
[0217] 5) The sample cartridge 200 is then removed from the Single
Sample Singulator instrument 2050, the nuclei 1050 suspension
pipetted into a 5 ml tube, and 2 mL of 4.degree. C. nuclei storage
buffer 413 added. The sample is then centrifuged at 4.degree. C.
for 5 minutes at 500 g. The supernatant is pipetted out and
discarded. The nuclei pellet is then resuspended in one mL of
nuclei storage buffer 413.
[0218] Example: Gene Expression Panels to Optimize the Performance
of Sissociation Methods.
[0219] Disrupting intact tissue into single cells can induce
transcriptional changes in the cells, through a process known as
anoikis or other stress-response pathways. Such changes can lead to
cell death or confound later genomic or proteomic analyses. Use of
quantitative PCR (qPCR assays) on a panel of targeted genes known
to be involved in anoikis or other cell-stress pathways can be used
to characterize the dissociation-related transcriptional changes in
the single cells produced by dissociation. The qPCR data can also
be used to inform and optimize the dissociation process to reduce
the stress-induced changes. While panels of genes have been
described for monitoring specific cell stress pathways, none have
been created to inform anoikis-induced stress or stress resulting
from mechanical and/or enzymatic/chemical tissue disruption.
[0220] qPCR panels can be used to identify specific cell types or
sub-cell types that are present in a mixture of dissociated cells
or characterize individual cells that have been isolated. The cell
identity information can in turn be used to inform and optimize the
dissociation process for desired cell types.
[0221] The panel may also be used to characterize RNA isolated from
nuclei as opposed to single cells. Processes for isolating nuclei
can be much faster than for isolating cells. The shorter process
time may reduce the amount of cell stress evident in the gene
expression data. In addition, isolated nuclei will lack RNA from
the cell cytoplasm and will therefore provide complementary data.
The qPCR data can also be used to inform and optimize the
dissociation process to reduce the stress-induced changes or to
identify specific cell types of origin for nuclei.
[0222] The structure of an exemplary panel for cell stress shown in
Table 1 is a collection of PCR primers chosen to amplify genes
associated with cell stress, and that have been optimized to
amplify RNA sequences rather than genomic DNA. The panels can
consists of 1 to over 200 genes, and may include at least one
housekeeping gene used as an internal control.
[0223] To use the panel, after isolation of cells from solid tissue
using a device such as, but not limited to, the Singulator System
100, a user would perform RT-qPCR on a panel of genes involved in
cell stress responses or cellular identity on a known number of
cells, or nuclei, or known quantity of isolated RNA. The levels of
gene expression would be determined and may be (1) compared to the
level of expression of so-called housekeeping genes whose
expression is not affected by cell stress responses, (2) compared
to the level of gene expression obtained from cells isolated using
a different isolation protocol, or (3) used to identify the
presence or absence or specific cell types. Other analyses are also
possible.
[0224] One example of a gene panel is shown below, with 38 genes
suspected of being involved in cellular stress responses and two
housekeeping genes used as internal controls. The genes have been
chosen because they are broadly expressed in most tissues, enabling
the panel to be used with cells derived from a variety of tissue
samples. The panel shown in Table 1 was developed for use with
mouse tissues. Genes marked with an asterix are the control
housekeeping genes.
[0225] As an example, a user would disrupt a fresh mouse liver
sample into a suspension of cells using the Singulator System 100
and a protocol for mouse liver. The user would then remove the cell
suspension 1000 from the Singulator sample cartridge 200. After
using a Countess, hemocytometer, FACS, or similar method to
determine cell concentration, the user could employ a "Cells to Ct"
kit (Invitrogen), or alternative method for performing cell lysis,
cDNA synthesis and qPCR with the primer sets for the genes listed
in the panel. Alternatively, the user could purify RNA from the
isolated cells using an RNA isolation kit, e.g., RNA Easy kit
(Qiagen), or alternatively, quantify the RNA concentration and
purity, then perform a cDNA synthesis and qPCR experiment using the
panel of genes. The qPCR amplification would be run on a Real Time
PCR instrument with a thermal cycling profile appropriate to the
kit or methods used. The qPCR experiment will return a cycle
threshold (Ct) values for each gene and these data can be used to
assess relative gene expression patterns.
[0226] Other assays for cell stress responses are available,
including those based on measuring apoptosis or necrosis of cells.
The panel defined in Table 1 is unique in that it represents genes
that span several cell stress pathways, whether known or
uncharacterized, and can be used to measure cell stress responses
specifically to a tissue disruption process. It is designed to
encompass an array of genes that may respond to tissue
dissociation, which may trigger multiple stress pathways, rather
than monitor one or a limited number of defined stress genes or
pathways.
[0227] Example: Determining the Extent and Specificity of Gene
Editing Methods
[0228] The use of CRISPR, TALENS, and other gene editing techniques
are being increasingly used to experimentally manipulate biological
systems for both research and clinical applications. Key metrics
for the success or failure of such manipulations are the number of
cells with effectively altered genomes and the specificity of such
alterations at the desired locations as opposed to off-target
editing. Off target gene editing can lead to disease-causing
changes to cells. It is difficult to assess the penetrance of
editing or the specificity through DNA or RNA sequencing of bulk
tissue, as rare events may not be observable. It is important to
perform an evaluation of gene editing using single cell sequencing
techniques.
[0229] In this example, a biological test subject, such as but not
limited to cell cultures, adherent cells, organoids, model
organisms, or human patients has been treated with a gene editing
process. Subsequent to the treatment, a sample or samples of cells
or tissue is removed from the test organism or culture media. The
sample, such as a tissue specimen 120 or microtissue 6001 or
organoid 6002, is processed in the Singulator 100 or alternative
device for tissue disruption to obtain a suspension of cells or
nuclei. The cells or nuclei are then subjected to single cell or
single nuclei DNA or RNA sequencing to determine the presence or
absence of an edited genome and the representation within the
single cell population.
[0230] As used herein, the following meanings apply unless
otherwise specified. The word "may" is used in a permissive sense
(i.e., meaning having the potential to), rather than the mandatory
sense (i.e., meaning must). The words "include", "including", and
"includes" and the like mean including, but not limited to. The
singular forms "a," "an," and "the" include plural referents. Thus,
for example, reference to "an element" includes a combination of
two or more elements, notwithstanding use of other terms and
phrases for one or more elements, such as "one or more." The term
"or" is, unless indicated otherwise, non-exclusive, i.e.,
encompassing both "and" and "or."
[0231] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of features is not necessarily limited only to those features
but may include other features not expressly listed or inherent to
such process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive-or
and not to an exclusive-or. For example, a condition A or B is
satisfied by any one of the following: A is true (or present) and B
is false (or not present), A is false (or not present) and B is
true (or present), and both A and B are true (or present). Both
plural and singular means may be included. The term "any of"
between a modifier and a sequence means that the modifier modifies
each member of the sequence. So, for example, the phrase "at least
any of 1, 2 or 3" means "at least 1, at least 2 or at least 3". The
term "consisting essentially of" refers to the inclusion of recited
elements and other elements that do not materially affect the basic
and novel characteristics of a claimed combination.
[0232] All patents, patent applications, published applications,
treatises and other publications referred to herein, both supra and
infra, are incorporated by reference in their entirety. If a
definition and/or description is set forth herein that is contrary
to or otherwise inconsistent with any definition set forth in the
patents, patent applications, published applications, and other
publications that are herein incorporated by reference, the
definition and/or description set forth herein prevails over the
definition that is incorporated by reference.
[0233] It should be understood that the description and the
drawings are not intended to limit the invention to the particular
form disclosed, but to the contrary, the intention is to cover all
modifications, equivalents, and alternatives falling within the
spirit and scope of the present invention as defined by the
appended claims. Further modifications and alternative embodiments
of various aspects of the invention will be apparent to those
skilled in the art in view of this description. Accordingly, this
description and the drawings are to be construed as illustrative
only and are for the purpose of teaching those skilled in the art
the general manner of carrying out the invention. It is to be
understood that the forms of the invention shown and described
herein are to be taken as examples of embodiments. Elements and
materials may be substituted for those illustrated and described
herein, parts and processes may be reversed or omitted, and certain
features of the invention may be utilized independently, all as
would be apparent to one skilled in the art after having the
benefit of this description of the invention. Changes may be made
in the elements described herein without departing from the spirit
and scope of the invention as described in the following claims.
Headings used herein are for organizational purposes only and are
not meant to be used to limit the scope of the description.
* * * * *
References