U.S. patent application number 13/847349 was filed with the patent office on 2013-11-28 for automated method and apparatus for embryonic stem cell culture.
This patent application is currently assigned to Cellular Dynamics International, Inc.. The applicant listed for this patent is Cellular Dynamics International, Inc.. Invention is credited to Nathaniel BEARDSLEY, Veit BERGENDAHL, Christine DAIGH, Megan FITZGERALD.
Application Number | 20130316445 13/847349 |
Document ID | / |
Family ID | 39672032 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316445 |
Kind Code |
A1 |
BEARDSLEY; Nathaniel ; et
al. |
November 28, 2013 |
AUTOMATED METHOD AND APPARATUS FOR EMBRYONIC STEM CELL CULTURE
Abstract
The invention concerns methods for automated culture of
embryonic stem cells (ESCs) such as human ESCs. In some aspects,
methods of the invention employ optimized culture media and limited
proteolytic treatment of cells to separate cell clusters for
expansion. Automated systems for passage and expansion of ESCs are
also provided.
Inventors: |
BEARDSLEY; Nathaniel;
(Oregon, WI) ; BERGENDAHL; Veit; (Madison, WI)
; FITZGERALD; Megan; (Madison, WI) ; DAIGH;
Christine; (Middletown, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cellular Dynamics International, Inc. |
Madison |
WI |
US |
|
|
Assignee: |
Cellular Dynamics International,
Inc.
Madison
WI
|
Family ID: |
39672032 |
Appl. No.: |
13/847349 |
Filed: |
March 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12164969 |
Jun 30, 2008 |
|
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13847349 |
|
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60947013 |
Jun 29, 2007 |
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Current U.S.
Class: |
435/303.3 ;
435/303.1 |
Current CPC
Class: |
C12N 2501/999 20130101;
C12N 2509/00 20130101; C12M 33/07 20130101; C12M 41/48 20130101;
C12M 23/50 20130101; C12N 5/0606 20130101; C12M 33/06 20130101;
C12N 2501/70 20130101 |
Class at
Publication: |
435/303.3 ;
435/303.1 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735 |
Goverment Interests
[0002] This invention was made with government support under
SBIR#0712181 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1-39. (canceled)
40. An apparatus for automated maintenance and expansion of
pluripotent cells comprising: a) an incubator; b) a liquid handler
unit; c) one or more reservoirs, said reservoirs comprising i) a
viable population of pluripotent cells, ii) a defined media, the
defined media being one in which pluripotent cells can be cultured
and maintained in an undifferentiated state, and iii) one or more
of a protease, protease inhibitor and a Rho-associated kinase
(ROCK) inhibitor; and d) a controller in communication with the
liquid handler unit, wherein the controller is configured to direct
the liquid handler unit to effect the automated expansion and
maintenance of the pluripotent cells.
41. The apparatus of claim 40, wherein the controller comprises a
computer-readable medium including an operating program.
42. The apparatus of claim 40, wherein the controller directs at
least one of: the liquid handler unit, the one or more reservoirs,
and the incubator to i) remove media from a pluripotent cell
population; ii) contact pluripotent cells with a proteolytic
enzyme; and iii) incubate pluripotent cells with a proteolytic
enzyme to separate cell clusters.
43. The apparatus of claim 40, wherein the apparatus further
comprises a mechanical agitator and aspirator, and the controller
further directs at least one of: the liquid handler unit, the one
or more reservoirs, and the incubator to subject the pluripotent
cells to mechanical agitation or aspiration to further separate
cell clusters.
44. The apparatus of claim 40, wherein the controller further
directs at least one of: the liquid handler unit, the one or more
reservoirs, and the incubator to contact incubated pluripotent
cells with a protease inhibitor.
45. The apparatus of claim 44, wherein the controller further
directs at least one of: the liquid handler unit, the one or more
reservoirs, and the incubator to contact incubated pluripotent
cells with a Rho-associated kinase (ROCK) inhibitor.
46. The apparatus of claim 40, wherein the liquid handler unit
comprises at least a first reservoir and a second reservoir,
wherein the first reservoir comprises a TeSR media, and wherein the
second reservoir comprises a TeSR media, a Rho-associated kinase
(ROCK) inhibitor, and a protease inhibitor.
47. The apparatus of claim 40, wherein the Rho-associated kinase
(ROCK) inhibitor is H-1152 or H-100.
48. The apparatus of claim 40, wherein the pluripotent cells are ES
cells or induced pluripotent cells (iPS).
49. The apparatus of claim 40, wherein the liquid handler comprises
a gripper tool and a liquid handling tool.
50. The apparatus of claim 40, wherein the pluripotent cells are
human ES cells.
51. The apparatus of claim 40, further comprising a robotic device
configured to facilitate fluid communication between the liquid
handler unit and the incubator.
52. The apparatus of claim 40, further comprising a second or more
reservoirs in communication with the liquid handler unit.
53. The apparatus of claim 52, wherein the second reservoir
comprises cell culture plates, cell growth media, or a proteolytic
enzyme solution.
54. The apparatus of claim 40, wherein the liquid handler is in
communication with at least a first reservoir and a second
reservoir, wherein the first reservoir comprises a TeSR media, and
wherein the second reservoir comprises a TeSR media which further
comprises a Rho-associated kinase (ROCK) inhibitor and a protease
inhibitor.
55. The apparatus of claim 40, wherein the controller is comprised
in a computer.
56. The apparatus of claim 40, wherein said incubator comprises a
portion of said pluripotent cells, and wherein the apparatus is
configured to provide for expansion of the pluripotent cells with
no or essentially no further differentiation of the cells in at
least 97% of the expanded pluripotent cells.
57. The apparatus of claim 40, wherein the incubator comprises a
portion of said pluripotent cells, and wherein the pluripotent
cells are human ES cells.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 12/164,969, filed on Jun. 30, 2008, which claims priority to
U.S. provisional Application No. 60/947,013 filed on Jun. 29, 2007.
The entire text of each of the above referenced disclosures is
specifically incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The instant invention concerns mammalian tissue cell culture
systems. More specifically, the invention concerns automated stem
cells culture systems.
[0005] 2. Description of Related Art
[0006] Since the inception of stable cultures of human embryonic
stem cells (ESCs) by Thomson et al. (1998) a growing number of
researchers have begun to explore possible therapeutic and
diagnostic uses for ESCs. However, even research use of ESCs has
strained the limited supplies of ESC cultures. Growing human ES
cells is a highly inefficient and variable process since culturing
techniques require a high degree of personnel skills and time.
Furthermore, the time, labor and complexity of ESC culture has
resulted in a very high cost for such cultures. Thus, current
methods for stem cell culture are inadequate even for production of
sufficient numbers of ECS to satisfy the demands of the research
community. Even greater numbers of ESCs will be required to
implement a commercially viable therapeutic and diagnostic use of
ES cells. Thus, there is need for improved cost effective methods
for culture of ESCs.
[0007] Previously, methods for automated maintenance of ES cell
cultures have been described (Terstegge et al., 2007); however,
such methods do not allow ES cell culture to be expanded and thus
fail to address the problems associated with large scale ES cell
production. Due to immense variability of manual procedures and
their limitations towards economical scale-up production of cells,
the ability to economically produce high quality cell lineages in
large quantities by automation will likely be a crucial criteria
that may define success in this very young and promising field.
Clearly, there exists a need for improved methods and systems for
culture and production of ES cells.
SUMMARY OF THE INVENTION
[0008] The present invention overcomes limitations in the prior art
by providing methods and compositions for the efficient passage and
expansion of embryonic stem cells. In particular embodiments, the
invention provides an optimized automated system for the efficient
culture and expansion of embryonic stem cells. For instance, in a
first embodiment there is provided a method for automated expansion
or passage of embryonic stem (ES) cells comprising (a) obtaining a
first population of ES cells in growth media, (b) separating the ES
cells with an automated separation system and (c) suspending the
separated cells in fresh growth media to provide an expanded
population of ES cells. In preferred aspects such methods may be
used for the passage or expansion of human embryonic stem cells
(hESCs). As used herein the term "passage" of cells refers to
culture of cells wherein the cells remain viable but may or may not
be actively dividing. Furthermore the term "expansion" refers to
growth of dividing cells wherein the number of cells increases with
culture time. Methods for obtaining embryonic stem cells, such as
human ESCs have been previously described for example see U.S. Pat.
Nos. 5,843,780, 6,200,806 and 7,029,913, each incorporated herein
by reference.
[0009] In certain aspects, the invention concerns cell growth
media. For example, in some aspects growth media may comprise
serum, such as human or bovine serum. In other aspects, growth
media may be defined as serum free media, serum protein free media
or protein free media. In various embodiments, no or essentially no
differentiation occurs in the cultured expanded ES cells; for
example, in the below examples at least 97% of the cultured
expanded ES cells remained in an undifferentiated state, based on
Oct4 expression. The skilled artisan will understand that media
according to the invention may comprise a number constituents
including but not limited to vitamins, buffers, glutamine, sugars
(e.g., pyruvate), reducing agents (e.g., beta mercaptoethanol),
antibiotics, antifungal agents, cytokines or growth factors.
Furthermore, in preferred aspects media for use according to the
invention may comprise components that reduce apoptosis in
disassociated ES cells. For example, media may comprise a
Rho-associated kinase (ROCK) inhibitor, such as Y-27632, HA-100,
H-1152 or a derivative thereof (Watanabe et al., 2007).
Furthermore, in certain aspects, a growth media according to the
invention may comprise an effective amount of a ROCK inhibitor,
such as an amount that is effective to prevent apoptosis in about
or more than about 50%, 60%, 70%, 80%, 90% or 95% of cells during
cell separation. In certain further aspects of the invention media
may be a "defined media" wherein the exact constituents of the
media formulation are known; for example, defined media do not
contain "undefined" animal products such as serum, which varies in
content between batches. In some very specific aspects, media for
use in the invention may be TeSR media, such as defined TeSR media
(Table 1; Ludwig & Thompson, 2007; Ludwig et al., 2006).
[0010] Various methods for culturing stem cells, e.g., human ESCs,
may be used with the present invention. Typically, ESCs are grown
in adherent culture systems such as on tissue culture plates. In
certain aspects, culture plates for use in the invention may
comprise a gel matrix such as a collagen or hydrogel matrix (e.g.,
a MATRIGEL.TM.). In various embodiments, culture plates may be
coated with, e.g., collagen IV, fibronectin, laminin, and
vitronectin in combination may be used to provide a solid support
for embryonic cell culturing and maintenance, as described in
Ludwig et al. (2006). Matrix components which may be used with the
present invention to coat tissue culture plates includes a collagen
such as collagen IV, laminin, vitronectin, Matrigel.TM., gelatin,
polylysine, thrombospondin (e.g., TSP-1, -2, -3, -4 and/or -5),
and/or ProNectin-F.TM.. Three dimensional support matrices for use
in tissue culture have been previously described for example in
U.S. Publication Nos. 20060198827 and 20060210596, each
incorporated herein by reference. The skilled artisan will
recognize that in certain aspects adherent tissue culture cells may
be defined by the cell density or confluency. Thus, in some cases,
methods of the invention involve expansion of proliferating cells
from a high density to a lower density to facilitate further cell
proliferation. For example, methods for expanding cells according
to the invention may involve a first population of ES cells that is
between about 50% and 99% confluent. For example, in certain
aspects the first population of ES cells may be about or less than
about 60%, 70%, 80%, 90% or 95% confluent. Furthermore, in certain
aspects expansion or passage of adherent ES cells may involve
seeding separated cells in fresh growth media. As used herein the
term "seeding" cells means dispersing cells in growth media such
that the resultant cell culture(s) are of approximately uniform
density. Thus, seeding of cells may involve mixing separated cells
with fresh growth media and/or spatially dispersing separated cells
over the surface of a tissue culture plate.
[0011] Furthermore, in certain aspects, methods of the invention
may involve seeding cells to a particular density in fresh media.
For example, in some cases, methods may be defined by the relative
density used for seeding of separated cells in fresh media. For
instance, separated cells may be seeded over a larger plate surface
area than the surface area that comprised the first population of
ES cells. In preferred aspects, the surface area of a new cell
culture plate(s) is about or between about 5 to about 35, between
about 10 and about 35, between about 15 and about 30, or between
about 28 and about 34, or about 30, 31, or 32 times greater than
the surface area of the plate comprising the first population of ES
cells. The skilled artisan will recognize that in certain aspects
expansion of cells according to the invention involves seeding
cells on larger culture plates, however in some cases cells may be
seeded on multiple plates wherein new plate surface area is defined
as the sum of the surface areas of the plates unto which the
separated cells are seeded. Thus, in some aspects, methods of the
invention may be used to produce a plurality of cell culture
populations from a starting cell culture population.
[0012] In some aspects the invention concerns a system for the
separation of cells, such as an automated separation system. In
certain aspects, ES cells may be mechanically or chemically
separated. Chemical separation may be achieved by using chelating
molecules (e.g., EDTA, EGTA, citrate or similar molecules that can
efficiently chelate or complex calcium and/or magnesium ions). In
other embodiments, urea may be used to separate or remove cells
from a cell culture plate. Removal of these ions distorts proteins
required for attachement of the cells to each other and to the
vessel surface. EDTA is present in most Trypsin reagens sold for
the purpose of detaching and individualizing cells. Chemicals may
be used in final concentrations of from about 0.01 mM to about 100
mM in the media to sufficiently break up and individualize the
cells. However, in certain cases, cells separation may be
facilitated by contacting the cells with an enzyme such as a
proteolytic enzyme. For example, a proteolytic enzyme may be
trypsin or typsin-like proteinase, such as purified or recombinant
proteinase. Thus, certain aspects enzymes for use according to the
invention may be recombinant enzymes that are essentially free from
other human or animal proteins or nucleic acids. In some specific
aspects a proteinase for use in the invention may be TRYPLE.TM..
Furthermore, in certain aspects, cells may be contacted with a
1.times. concentration of TRYPLE.TM. enzyme solution. The skilled
artisan will recognize that concentration of an enzyme used in
methods of the invention will depend upon the length of time cells
are exposed to the enzyme (i.e., the time cells are exposed to the
active enzyme) and the temperature during exposure/incubation.
Furthermore, proteins in culture media can reduce the efficacy
proteolytic enzymes in separation of cell clusters thus, in certain
aspects a cell growth media may be removed prior to contacting
cells with a proteolytic enzyme. Thus, in certain aspects, methods
of the invention may comprise a system for cell separation
comprising (i) removing the media from the first ES cell
population, (ii) contacting the ES cell population with a
proteolytic enzyme, and (iii) incubating the cell population with a
proteolytic enzyme to separate cell clusters. For example, in some
cases ES cells are incubated with the proteolytic enzyme or
chemical for between about 2 and about 10 minutes such as for about
or at most about 3, 4, 5, 6, 7, 8 or 9 minutes. The skilled artisan
will also recognize that enzymatic activity is typically
temperature dependent thus activity may be modulated by changing
the incubation temperature. Thus, in certain aspects, ES cells may
be incubated with an enzyme such as trypsin at between about
25.degree. C. and about 40.degree. C., such as at about 26.degree.
C., 27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
31.degree. C., 32.degree. C., 33.degree. C., 34.degree. C.,
35.degree. C., 36.degree. C., 37.degree. C., 38.degree. C. or
39.degree. C.
[0013] The skilled artisan will recognize that excessive exposure
of cells to a proteolytic enzyme such as trypsin can be detrimental
to cell viability. Thus, in certain aspects, proteinase incubation
may be monitored to determine the length of the incubation that is
required to separate cells from a tissue culture plate or to
separate cell clusters. For example, incubation may be monitored
via microscopy or by flow cytometry (e.g., to assess the size of
cell clusters). Methods for performing flow cytometry are well
known in the art see for example, U.S. Pat. Nos. 4,284,412,
4,989,977, 4,498,766, 5,478,722, 4,857,451, 4,774,189, 4,767,206,
4,714,682, 5,160,974, and 4,661,913. In some preferred aspects
proteinase incubation may be monitored by a computer and the
incubation may be halted (e.g., by addition of a proteinase
inhibitor) when optimal cell separation is achieved. Thus, in some
cases fresh media for cell dilution or seeding may comprise an
enzyme inhibitor, such a protease inhibitor. In certain aspects
fresh media for use according to the invention may comprise an
inhibitor of the proteolytic enzyme used for cell separation. For
example, in preferred aspects, fresh media may comprise an amount
of an enzyme inhibitor sufficient to inhibit about or at least
about 70%, 80%, 90%, 95%, 98%, 99% or substantially all of enzyme
activity. For example, in the case where trypsin is used in cell
separation system according to the invention, fresh media for may
comprise a trypsin inhibitor such as soybean trypsin inhibitor. For
instance, in some very specific aspects, fresh growth media may
comprise about 0.5 mg/ml of soybean trypsin inhibitor. In other
embodiments natural trypsin inhibitors, such as the ones present in
serum may be used with the present invention, e.g., to be included
in a media during the splitting of cells. In still further aspects,
cell growth media may be further replaced with a media that does
not comprise an enzyme inhibitor after the enzyme has been
essentially inactivated.
[0014] Various other protease inhibitors may be used with the
present invention. In most cases dilution of the proteolytic enzyme
is sufficient to prevent damage to the cells. Non-limiting examples
of protease inhibitors that may be used with the present invention
include may be obtained from: serum (e.g., .alpha.1-antitrypsin, a
.about.52 kDa serum trypsin inhibitor), lima beans (e.g., six lima
bean inhibitors are known which are .about.8-10 kDa), bovine
pancreas (e.g., Kunitz inhibitor, also known as aprotinin,
.about.6.5 kDa), avian egg whites (e.g., ovomucoids are
glycoprotein protease inhibitors found in avian egg white,
.about.8-10 kDa), and/or soybeans (several inhibitors are known,
typically .about.20.7-22.3 kDa).
[0015] In certain preferred aspects, methods according to the
invention may be automated. For example, a liquid handler robot may
be used to automate the methods described herein. A wide array of
liquid handler robots are known in the art and may be used
according to the invention, for example see U.S. Pat. No.
6,325,114, incorporated herein by reference in its entirety. In
some aspects, a robot for use according to the invention may be a
Beckman Coulter BIOMEK.RTM. 2000 liquid handler (B2K). Furthermore,
it is contemplated that an automated system or apparatus for use
according to the invention may comprise a bioreactor wherein fluid
transfer and/or cell seeding is mediated by pumps or pressure
gradients. As shown in the below examples, an automated apparatus
and system was produced for feeding and reproducibly splitting
human ESCs; the human ESC's cultured using this apparatus and
system were of high quality and did not display significant
differentiation (greater than 97% undifferentiated, as measured by
Oct4 FACS analysis). While the specific cells used in the below
examples were human ESC's, the inventors anticipate that other
human or mammalian stem cells or iPS cells may be cultured,
expanded, and maintained in an undifferentiated state according to
the present invention.
[0016] In various embodiments, the methods of the present invention
may be used to screen compounds which may modulate the
differentiation state of a cell. As shown in the below examples,
the inventors have demonstrated successfully that this technology
may be used for human ESC cell culture-based small molecule
screening using defined culture conditions. In certain embodiments,
the methods, apparatus and systems of the present invention may be
used to screen one or more candidate compound(s) which may affect
the differentiation state of a cell. For example, the candidate
compound may promote differentiation of a stem cell towards a
specific lineage (e.g., hematopoietic, etc.). In other embodiments,
the candidate compound may promote de-differentiation or maintain a
de-differentiated state in a cell (e.g., promote the generation of
an iPS cell from a fibroblast or other cell).
[0017] In still further aspects, methods of the invention may
comprise an apparatus or system for separating cells comprising a
combination or mechanical separation and enzymatic separation. For
example, in some cases, cells may be incubated with an enzyme such
as trypsin followed by mechanical agitation to further separate
cell clusters. For example, mechanical agitation may comprise
subjecting cells to shear forces, such as by pipetting the cells
repeatedly through an aperture.
[0018] The skilled artisan will recognize that a number of ES cell
culture system comprises "feeder cells" that supply, in trans,
factors that mediate ES cell growth and/or differentiation.
However, in certain aspects, methods of the invention concern a
population of ES cells that is essentially free from non-ES cells
or essentially free from non-human cells.
[0019] In still further embodiments, a method of the invention may
be defined as an automated method for serial expansion of embryonic
stem (ES) cells comprising (a) obtaining a first population of ES
cells in growth media, (b) separating the ES cells with an
automated separation system, (c) suspending the separated cells in
fresh growth media to provide an expanded population of ES cells,
(d) incubating the expanded ES cell population under conditions
supporting cell growth and (e) repeating steps b-d one or ore times
to provide a serially expanded population of ES cells. Thus,
methods of the invention may be used for the passage or expansion
of a population of stem cells for any number of passages from
initial ES culture to senescence of the cells.
[0020] In still a further embodiment of the invention there is
provided a system for automated expansion of ES cells comprising an
incubator, a liquid handler unit and an operating program for cell
separation. For example, an operating program may comprise steps
for (i) removing media from a first ES cell population, (ii)
contacting ES cells with a proteolytic enzyme, (iii) incubating the
cells with a proteolytic enzyme to separate cell clusters and/or
(iv) subjecting the incubated cells to mechanical agitation to
further separate cell clusters. Thus, in some aspects, an operating
program may be used to move cells and or fluids between different
chambers in the system. In some aspects, cell culture plates may be
moved for one chamber to another (e.g., into or out of an
incubator). Thus, in certain aspects, a liquid handler may comprise
a gripper tool and a liquid handling tool. In still further aspects
a liquid handling tool may be an essentially closed system or
apparatus wherein cells and/or fluids are moved between chambers by
a pressure gradient.
[0021] It is anticipated that virtually any pluripotent stem cell
or cell line, e.g., human embryonic stem cells or induced
pluripotent stem cells (iPS cells), may be cultured via the present
invention. For example, human embryonic stem cell line H1, H9,
hES2, hES3, hES4, hES5, hES6, BG01, BG02, BG03, HSF1, HSF6, H1, H7,
H9, H13B, and/or H14 etc. may be used with the present invention.
It is further anticipated that stem cell lines which subsequently
become available may also be used with the present invention.
Although human embryonic stem cells are preferably used with the
present invention, in some instances it may also be possible to use
other embryonic stem cells, such as mammal, mouse, primate, etc.
with the present invention.
[0022] As would be appreciated by one of skill, induced pluripotent
stem cells, commonly abbreviated as iPS cells or iPSCs, are a type
of pluripotent stem cell artificially derived from a
non-pluripotent cell, typically an adult somatic cell, by inserting
certain genes. Induced pluripotent stem cells are believed to be
identical to natural pluripotent stem cells, such as embryonic stem
cells in many respects, such as in terms of the expression of
certain stem cell genes and proteins, chromatin methylation
patterns, doubling time, embryoid body formation, teratoma
formation, viable chimera formation, and potency and
differentiability, but the full extent of their relation to natural
pluripotent stem cells is still being assessed. IPS cells have been
described previously (see, e.g., Takahashi et al., 2006; Takahashi
et al., 2007; Yu et al, 2007).
[0023] Embodiments discussed in the context of a methods and/or
composition of the invention may be employed with respect to any
other method or composition described herein. Thus, an embodiment
pertaining to one method or composition may be applied to other
methods and compositions of the invention as well.
[0024] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0025] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0026] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0027] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following drawing is part of the present specification
and is included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to the drawings in combination with the detailed
description of specific embodiments presented herein.
[0029] FIG. 1: An example automated method of embryonic stem
expansion. One or more of the depicted steps may be comprised in a
program to control a system for stem cell expansion.
[0030] FIGS. 2A-B: Diagrams of exemplary apparatus and systems for
automated expansion of ES cells.
[0031] FIGS. 3A-B depict an example of an automated human ES (HES)
culturing apparatus and system in a clean room. FIG. 3A, depicts an
example of an open automated incubator (Cytomat 6000) next to the
Platecrane XT (Hudson) connecting the liquid handler (Biomek2000).
FIG. 3B depicts an example of the closed Cytomat 6000 on the left
side of the system, the Biomek2000 from the back in the middle, and
below a temperature control unit. The Stacker of the Biomek2000
system is placed on the right side of the system. FIGS. 3C-D depict
robotic components for splitting and feeding HES cells. FIG. 3C
depicts a single channel wash tool used initially to aspirate spent
media and feed 4-well or Swell plates. In the back behind the
4-well plate a 6-well plate is used to serve as a reservoir for
trypsin. FIG. 3D depicts an 8-channel tool (P200) used in certain
examples to mix and dispense the trypsinized and individualized HES
cells from a 4-well mother plate to an 8-well daughter plate in the
final split.
[0032] FIGS. 4A-C: Phase contrast microscopy pictures of human ES
cells after feeding. FIG. 4A, 20.times. magnification of a single
2-days old human ES cell colony. FIG. 4B, 4.times. magnification of
a 2-days old culture to show the distribution and density of the
cultured cells. FIG. 4C, A H1 culture after 5 days of feeding at
4.times. magnification.
[0033] FIGS. 5A-B: Phase contrast microscopy picture of human ES
cells after 1000-fold expansion. FIG. 5A, 4.times. magnification of
human ES cell colonies 6-days after the 2nd passage in the
scale-up. FIG. 5B, Oct4 analysis by FACS. The left band shows cells
treated with the labeled IgG control to show the background and the
solid band represents the Oct4 positive population from plate 2
pooled and stained. Both analyses showed greater than 97% Oct4
positive population, suggesting that the automated procedure
effectively maintains pluripotent cells in an undifferentiated
state.
[0034] FIG. 6: Cell counts from randomly picked plates after
1000-fold expansion using the automated cell culture system. Plates
were trypsinized and stained with trypan blue and counted on a
hemacytometer. Total cells were calculated from representative
samples. Extrapolated from the mean value indicates that
approximately 160.times.16 million=2.56 billion human ES cells were
generated.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Human stem cells are currently being developed for use in a
variety of therapeutic and diagnostic applications. In particular,
ESCs maybe differentiated into a variety of cell types and thus may
be used to treat or study disease of a variety of human tissues.
However, availability of large numbers of cultured human stem cells
has proven to be a major limitation in the field. Unlike
conventional tissue culture of transformed cell lines, ESCs are
very sensitive to growth conditions and the surrounding
microenvironment can modulate the cell viability and the speed at
which ESCs proliferate. Furthermore, ESC culture is very human
labor intensive thereby increasing the cost of expanding cell
populations and increasing the probability of contamination of cell
cultures. Even these laborious methods of cell culture have
typically only enabled about a 1:12 expansion ratio, thereby
limiting the number of cells that could be grown over a particular
period of time and increasing the frequency of cell splitting
required to maintain maximal cell proliferation rates.
[0036] The instant invention addresses many of the deficiencies of
previous methods for ESC culture in providing an automated method
for passaging and expanding ESC cell cultures. As shown in the
below examples, an apparatus and system was produced which allowed
for the automated feeding and splitting of ES cells which allowed
for the expansion of one well of a 6-well plate of HES cells (-2.5
million cells) to a final number of 160 plates (.about.2-3 billion
cells). This is the equivalent to a 1000-fold expansion over 3
weeks, an otherwise nearly impossible task for a single person. As
shown by Oct4 staining, the vast majority of these stem cells,
i.e., greater than 97%, remained in an undifferentiated state.
[0037] The instant invention provided an automated ESC culture
system that employs a limited enzymatic treatment of cell clusters
to separate the cells for seeding on new plates. Thus, in some
aspects mechanical agitation of cell cultures is limited and a
larger portion of viable ESCs are carried for passage to passage.
In particular, methods and compositions provided herein enabled
cells to be expanded from one plate to 30 plates (i.e., to a
30.times. greater surface area) in a single split, which is an
improvement over hand-splitting methods, which typically allow for
no more than 12-fold expansion at any given time. Furthermore,
automated systems described here greatly reduce the need for human
labor and thus the cost of culturing cells. Such automated
apparatus and systems may be less prone to contamination and are
preferred for stem cell products that may be ultimately used as
therapeutics. Thus, the instant invention may enable rapid
commercial development of ESC therapeutics such as, e.g., ESC
derived blood for use in transfusion.
I. Cell Growth Media
[0038] A variety of media an culture conditions for ES cell culture
are known in the art. In certain aspects, cells may be grown with
feeder cells such a fibroblasts or in fibroblast conditioned media.
However, in some instances it may be preferred that ES cells are
grown in the absence of feeder cells. In still more preferred
aspects cells may be grown in a defined media such as TeSR (e.g.,
MTESR.TM.1 available from BD Biosciences) (Ludwig et al., 2006a,
U.S. Application 2006/0084168). Such media may be used for serum
free culture of ES cells. For example, in some cases growth media
may be the media defined in Table 1. However, due to the high cost
of serum free systems in certain cases growth factors used for
serum free culture may be obtained from alternate sources to reduce
cost, such a FGF cloned from zebra fish as described by Ludwig et
al. (2006b). Furthermore, in certain aspects, media is supplemented
with bovine or human serum to supply the necessary growth factors
(Ludwig et al., 2006b). Thus, in certain cases, an ES growth media
may comprise the ingredients as shown in Table 1, wherein the media
is supplemented with bovine serum in place of the indicated "growth
factors and proteins," as exemplified herein.
TABLE-US-00001 TABLE 1 Formulation for TeSR1 Medium mM mM INORGANIC
SALTS AMINO ACIDS Calcium chloride (Anhydrous) 8.24E-01 L-Alanine
1.37E-01 HEPES 1.18E+01 L-Arginine hydrochloride 5.48E-01 Lithium
Chloride (LiCl) 9.80E-01 L-Asparagine-H.sub.2O 1.37E-01 Magnesium
chloride (Anhydrous) 2.37E-01 L-Aspartic acid 1.37E-01 Magnesium
Sulfate (MgSO.sub.4) 3.19E-01 L-Cysteine-HCl--H.sub.2O 7.83E-02
Potassium chloride (KCl) 3.26E+00 L-Cystine 2HCl 7.83E-02 Sodium
bicarbonate (NaHCO.sub.3) 1.80E+01 L-Glutamic acid 1.37E-01 Sodium
chloride (NaCl) 9.46E+01 L-Glutamine 2.94E+00 Sodium phosphate,
dibas (Anhydrous) 3.92E-01 Glycine 2.94E-01 Sodium phosphate,
3.55E-01 L-Histidine-HCl--H.sub.2O 1.18E-01 mono. L-Isoleucine
3.26E-01 (NaH.sub.2PO.sub.4--H20) L-Leucine 3.54E-01 TRACE MINERALS
L-Lysine hydrochloride 3.91E-01 Ferric Nitrate
(Fe(NO.sub.3).sub.3--9H.sub.2O) 9.71E-05 L-Methionine 9.06E-02
Ferric sulfate (FeSO.sub.4--7H.sub.2O) 1.18E-03 L-Phenylalanine
1.69E-01 Cupric sulfate (CuSO.sub.4--5H.sub.2O) 4.08E-06 L-Proline
2.16E-01 Zinc sulfate (ZnSO.sub.4--7H.sub.2O) 1.18E-03 L-Serine
2.94E-01 Ammonium Metavanadate NH.sub.4VO.sub.3 1.09E-05
L-Threonine 3.52E-01 Mangenous Sulfate Mn SO.sub.4 H.sub.2O
1.97E-06 L-Tryptophan 3.46E-02 NiSO.sub.4 6H.sub.2O 9.70E-07
L-Tyrosine 1.68E-01 Selenium 1.77E-04 2Na 2H.sub.2O Sodium Meta
Silicate Na.sub.2SiO.sub.3 9H.sub.2O 9.66E-04 L-Valine 3.55E-01
SnCl.sub.2 1.24E-06 VITAMINS Molybdic Acid, Ammonium salt 1.97E-06
Ascorbic acid 2.53E-01 CdCl.sub.2 1.22E-05 Biotin 1.12E-05
CrCl.sub.3 1.98E-06 B12 3.94E-04 AgNO.sub.3 9.81E-07 Choline
chloride 5.03E-02 AlCl.sub.3 6H.sub.2O 4.87E-06 D-Calcium
pantothenate 3.69E-03 Ba (C.sub.2H.sub.3O.sub.2).sub.2 9.79E-06
Folic acid 4.71E-03 CoCl.sub.2 6H.sub.2O 9.81E-06 i-Inositol
5.49E-02 GeO.sub.2 4.97E-06 Niacinamide 1.30E-02 KBr 9.89E-07
Pyridoxine hydrochloride 7.62E-03 KI 1.00E-06 Riboflavin 4.56E-04
NaF 9.83E-05 Thiamine 2.42E-02 RbCl 9.81E-06 hydrochloride
ZrOCl.sub.2 9.80E-06 GROWTH FACTORS/PROTEINS 8H.sub.2O GABA
9.79E-01 ENERGY SUBSTRATES Pipecolic Acid 9.84E-04 D-Glucose
1.37E+01 bFGF 5.77E-06 Sodium 3.92E-01 TGF beta 1 2.35E-08 Pyruvate
Human Insulin 3.92E-03 LIPIDS Human Holo-Transferrin 1.37E-04
Linoleic Acid 1.88E-04 Human Serum Albumin 1.95E-01 Lipoic Acid
4.00E-04 Glutathione 6.38E-03 Arachidonic Acid 1.29E-05 (reduced)
Cholesterol 1.12E-03 OTHER COMPONENTS DL-alpha tocopherol-acetate
2.90E-04 Hypoxanthine Na 1.18E-02 Linolenic Acid 6.99E-05 Phenol
red 1.69E-02 Myristic Acid 8.59E-05 Putrescine-2HCl 3.95E-04 LIPIDS
OTHER COMPONENTS Oleic Acid 6.94E-05 Thymidine 1.18E-03 Palmitic
Acid 7.65E-05 2-mercaptoethanol 9.80E-02 Palmitoleic acid 7.71E-05
Pluronic F-68 2.33E-02 Stearic Acid 6.89E-05 Tween 80 3.29E-04
[0039] A. ROCK Inhibitors
[0040] In still further aspects of the invention additional media
components may be included in ES cell growth media such as
molecules that reduce ES cell apoptosis when cells become
disassociated (e.g., during splitting of cell populations). For
example, media for use in the invention may comprise one or more
Rho-associated kinase (ROCK) inhibitor such a Y-27632 or a
derivative thereof. Furthermore, in some aspects, media of the
invention may comprise HA-100: or a derivative thereof.
##STR00001##
[0041] The HA-100 may be present in an ES cell growth media, e.g.,
at a concentration of about 1-15 .mu.M, 5-15 .mu.M, 1-30 .mu.M,
5-30 .mu.M, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 .mu.M, or any
range derivable therein. In certain embodiments HA-100 is present
in an ES cell growth media at about 10-20 .mu.M.
[0042] Other ROCK inhibitors which may be included in an ES cell
growth media according to the present invention include H-1152
((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine).
H-1152 exhibits an approximately ten-fold greater potency than
HA-100. Thus, H-1152 may be present in an ES cell growth media,
e.g., at a concentration of about 0.1-10 .mu.M, about 0.5-5 .mu.M,
about 1-3 .mu.M, or about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5
.mu.M, or any range derivable therein. In certain embodiments
HA-100 is present in an ES cell growth media at about 1 .mu.M.
H-1152, which allows for very efficient seeding of individualized
human ES cells in 96-well plates (similar to HA-100 but at 10-fold
lower concentration). Individualized HES cells that are otherwise
passaged in cell clumps allow more uniform cell densities per well,
which is a stringent prerequisite for cell-based small molecule
screening. H-1152 can thus be used in protocols for ES cell-based
small molecule screening which involve automated cell culture
according to the present invention. H-1152 has been previously
described in, e.g., Ikenoya et al. (2002) and Sasaki et al. (2002),
which are incorporated herein by reference.
##STR00002##
[0043] Other ROCK inhibitors which may be included in an ES cell
growth media include Y-27632,
N-(4-Pyridyl)-N'-(2,4,6-trichlorophenyl)urea,
3-(4-Pyridyl)-1H-indole, glycyl-H1152
((S)-(+)-2-Methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopipera-
zine) and/or HA1100 (Hydroxyfausdil). Y-27632
((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide)
is commercially available from Sigma-Aldrich and has been described
previously (see, e.g., Maekawa et al., 1999; Davies et al.,
2000).
##STR00003##
II. Cell Culture Apparatus, Systems and Methods
[0044] In some aspects, the present invention may take advantage of
bioreactor technology. Growing cells according to the present
invention in a bioreactor allows for large scale production of
fully biologically-active cells capable of further differentiation
for end use. Bioreactors have been widely used for the production
of biological products from both suspension and anchorage dependent
animal cell cultures. Microcarrier cell culture in stirred tank
bioreactor provides very high volume-specific culture surface area
and has been used for the production of viral vaccines (Griffiths,
1986). Furthermore, stirred tank bioreactors have industrially been
proven to be scaleable, however such technologies may only be
employed when cells may be grown in anchorage independent cultures.
The multiplate CELLCUBE.TM. cell culture system manufactured by
Corning-Costar also offers a very high volume-specific culture
surface area. Cells grow on both sides of the culture plates
hermetically sealed together in the shape of a compact cube. Unlike
stirred tank bioreactors, the CELLCUBE.TM. culture unit is
disposable. This is very desirable at the early stage production of
clinical product because of the reduced capital expenditure,
quality control and quality assurance costs associated with
disposable systems.
[0045] A. Non-Perfused Attachment Systems
[0046] Traditionally, anchorage-dependent cell cultures are
propagated on the bottom of small glass or plastic vessels as
described herein. The restricted surface-to-volume ratio offered by
classical and traditional techniques, suitable for the laboratory
scale, has created a bottleneck in the production of cells and cell
products on a large scale. In an attempt to provide systems that
offer large accessible surfaces for cell growth in small culture
volume, a number of techniques have been proposed: the roller
bottle system, the stack plates propagator, the spiral film bottle
system, the hollow fiber system, the packed bed, the plate
exchanger system, and the membrane tubing reel. Since these systems
are non-homogeneous in their nature, and are sometimes based on
multiple processes, they suffer from the following
shortcomings--limited potential for scale-up, difficulties in
taking cell samples, limited potential for measuring and
controlling key process parameters and difficulty in maintaining
homogeneous environmental conditions throughout the culture.
[0047] Despite these drawbacks, a commonly used process for large
scale anchorage-dependent cell production is the roller bottle.
Being little more than a large, differently shaped T-flask,
simplicity of the system makes it very dependable and, hence,
attractive. Fully automated robots are available that can handle
thousands of roller bottles per day, thus eliminating the risk of
contamination and inconsistency associated with the otherwise
required intense human handling.
[0048] B. Cultures on Microcarriers
[0049] In an effort to overcome the shortcomings of the traditional
anchorage-dependent culture processes, van Wezel (1967) developed
the concept of the microcarrier culturing systems. In this system,
cells are propagated on the surface of small solid particles
suspended in the growth medium by slow agitation. Cells attach to
the microcarriers and grow gradually to confluency on the
microcarrier surface. In fact, this large scale culture system
upgrades the attachment dependent culture from a single disc
process to a unit process in which both monolayer and suspension
culture have been brought together. Thus, combining the necessary
surface for a cell to grow with the advantages of the homogeneous
suspension culture increases production.
[0050] The advantages of microcarrier cultures over most other
anchorage-dependent, large-scale cultivation methods are several
fold. First, microcarrier cultures offer a high surface-to-volume
ratio (variable by changing the carrier concentration) which leads
to high cell density yields and a potential for obtaining highly
concentrated cell products. Cell yields are up to
1-2.times.10.sup.7 cells/ml when cultures are propagated in a
perfused reactor mode. Second, cells can be propagated in one unit
process vessels instead of using many small low-productivity
vessels (i.e., flasks or dishes). This results in far better
nutrient utilization and a considerable saving of culture medium.
Moreover, propagation in a single reactor leads to reduction in
need for facility space and in the number of handling steps
required per cell, thus reducing labor cost and risk of
contamination. Third, the well-mixed and homogeneous microcarrier
suspension culture makes it possible to monitor and control
environmental conditions (e.g., pH, pO.sub.2, and concentration of
medium components), thus leading to more reproducible cell
propagation and product recovery. Fourth, it is possible to take a
representative sample for microscopic observation, chemical
testing, or enumeration. Fifth, since microcarriers settle out of
suspension quickly, use of a fed-batch process or harvesting of
cells can be done relatively easily. Sixth, the mode of the
anchorage-dependent culture propagation on the microcarriers makes
it possible to use this system for other cellular manipulations,
such as cell transfer without the use of proteolytic enzymes,
cocultivation of cells, transplantation into animals, and perfusion
of the culture using decanters, columns, fluidized beds, or hollow
fibers for microcarrier retainment. Seventh, microcarrier cultures
are relatively easily scaled up using conventional equipment used
for cultivation of microbial and animal cells in suspension.
[0051] C. Microencapsulation of Mammalian Cells
[0052] One method which has shown to be particularly useful for
culturing mammalian cells is microencapsulation. The mammalian
cells are retained inside a semipermeable hydrogel membrane. A
porous membrane is formed around the cells permitting the exchange
of nutrients, gases, and metabolic products with the bulk medium
surrounding the capsule. Several methods have been developed that
are gentle, rapid and non-toxic and where the resulting membrane is
sufficiently porous and strong to sustain the growing cell mass
throughout the term of the culture. These methods are all based on
soluble alginate gelled by droplet contact with a
calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,
incorporated herein by reference,) describes cells concentrated in
an approximately 1% solution of sodium alginate which are forced
through a small orifice, forming droplets, and breaking free into
an approximately 1% calcium chloride solution. The droplets are
then cast in a layer of polyamino acid that ionically bonds to the
surface alginate. Finally the alginate is reliquefied by treating
the droplet in a chelating agent to remove the calcium ions. Other
methods use cells in a calcium solution to be dropped into a
alginate solution, thus creating a hollow alginate sphere. A
similar approach involves cells in a chitosan solution dropped into
alginate, also creating hollow spheres.
[0053] Microencapsulated cells are easily propagated in stirred
tank reactors and, with beads sizes in the range of 150-1500 .mu.m
in diameter, are easily retained in a perfused reactor using a
fine-meshed screen. The ratio of capsule volume to total media
volume can be maintained from as dense as 1:2 to 1:10. With
intracapsular cell densities of up to 10.sup.8, the effective cell
density in the culture is 1-5.times.10.sup.7.
[0054] The advantages of microencapsulation over other processes
include the protection from the deleterious effects of shear
stresses which occur from sparging and agitation, the ability to
easily retain beads for the purpose of using perfused systems,
scale up is relatively straightforward and the ability to use the
beads for implantation.
[0055] D. Perfused Attachment Systems
[0056] Perfused attachment systems are a preferred form of the
present invention. Perfusion refers to continuous flow at a steady
rate, through or over a population of cells (of a physiological
nutrient solution). It implies the retention of the cells within
the culture unit as opposed to continuous-flow culture which washes
the cells out with the withdrawn media (e.g., chemostat). The idea
of perfusion has been known since the beginning of the century, and
has been applied to keep small pieces of tissue viable for extended
microscopic observation. The technique was initiated to mimic the
cells milieu in vivo where cells are continuously supplied with
blood, lymph, or other body fluids. Without perfusion, cells in
culture go through alternating phases of being fed and starved,
thus limiting full expression of their growth and metabolic
potential.
[0057] The current use of perfused culture is in response to the
challenge of growing cells at high densities (i.e.,
0.1-5.times.10.sup.8 cells/ml). In order to increase densities
beyond 2-4.times.10.sup.6 cells/ml, the medium has to be constantly
replaced with a fresh supply in order to make up for nutritional
deficiencies and to remove toxic products. Perfusion allows for a
far better control of the culture environment (pH, pO.sub.2,
nutrient levels, etc.) and is a means of significantly increasing
the utilization of the surface area within a culture for cell
attachment.
[0058] The development of a perfused packed-bed reactor using a bed
matrix of a non-woven fabric has provided a means for maintaining a
perfusion culture at densities exceeding 10.sup.8 cells/ml of the
bed volume (CELLIGEN, New Brunswick Scientific, Edison, N.J.; Wang
et al., 1992; Wang et al., 1993; Wang et al., 1994). Briefly
described, this reactor comprises an improved reactor for culturing
of both anchorage- and non-anchorage-dependent cells. The reactor
is designed as a packed bed with a means to provide internal
recirculation. Preferably, a fiber matrix carrier is placed in a
basket within the reactor vessel. A top and bottom portion of the
basket has holes, allowing the medium to flow through the basket. A
specially designed impeller provides recirculation of the medium
through the space occupied by the fiber matrix for assuring a
uniform supply of nutrient and the removal of wastes. This
simultaneously assures that a negligible amount of the total cell
mass is suspended in the medium. The combination of the basket and
the recirculation also provides a bubble-free flow of oxygenated
medium through the fiber matrix. The fiber matrix is a non-woven
fabric having a "pore" diameter of from 10 .mu.m to 100 .mu.m,
providing for a high internal volume with pore volumes
corresponding to 1 to 20 times the volumes of individual cells.
[0059] In comparison to other culturing systems, this approach
offers several significant advantages. With a fiber matrix carrier,
the cells are protected against mechanical stress from agitation
and foaming. The free medium flow through the basket provides the
cells with optimum regulated levels of oxygen, pH, and nutrients.
Products can be continuously removed from the culture and the
harvested products are free of cells and can be produced in
low-protein medium which facilitates subsequent purification steps.
Also, the unique design of this reactor system offers an easier way
to scale up the reactor. Currently, sizes up to 30 liter are
available. One hundred liter and 300 liter versions are in
development and theoretical calculations support up to a 1000 liter
reactor. This technology is explained in detail in WO 94/17178
(Aug. 4, 1994, Freedman et al.), which is hereby incorporated by
reference in its entirety.
[0060] The CELLCUBE.TM. (Corning-Costar) module provides a large
styrenic surface area for the immobilization and growth of
substrate attached cells. It is an integrally encapsulated sterile
single-use device that has a series of parallel culture plate
joined to create thin sealed laminar flow spaces between adjacent
plates.
[0061] The CELLCUBE.TM. module has inlet and outlet ports that are
diagonally opposite each other and help regulate the flow of media.
During the first few days of growth the culture is generally
satisfied by the media contained within the system after initial
seeding. The amount of time between the initial seeding and the
start of the media perfusion is dependent on the density of cells
in the seeding inoculum and the cell growth rate. The measurement
of nutrient concentration in the circulating media is a good
indicator of the status of the culture. When establishing a
procedure it may be necessary to monitor the nutrients composition
at a variety of different perfusion rates to determine the most
economical and productive operating parameters.
[0062] Cells within the system reach a higher density of solution
(cells/ml) than in traditional culture systems. Many typically used
basal media are designed to support 1-2.times.10.sup.6
cells/ml/day. A typical CELLCUBE.TM., run with an 85,000 cm.sup.2
surface, contains approximately 6 L media within the module. The
cell density often exceeds 10.sup.7 cells/mL in the culture vessel.
At confluence, 2-4 reactor volumes of media are required per
day.
III. Apparatus/Systems for Automated Expansion of ES Cells
[0063] Certain aspects of the invention concern apparatus or
systems for automated expansion of pluripotent cells, such as ES
cells, depicted in diagram form in FIGS. 2A and 2B, and illustrated
with commonly available hardware elements in FIGS. 3A-D. Thus, as
can be seen, an exemplary device can comprise a viable ES cell
population (102), a liquid handler unit (100) in fluid
communication with an incubator (104) and a controller (106)
comprising an operating program for cell separation.
[0064] ES cell populations (102) for use in an apparatus of the
invention may comprise an ES cell population from any source known
to those of skill in the art. For instance, methods for obtaining
embryonic stem cells, such as human ESCs have been previously
described in U.S. Pat. Nos. 5,843,780, 6,200,806 and 7,029,913. It
is understood that the term apparatus as used herein is not limited
to devices in a single housing, and may include mulitple devices
linked together, for example, via electrical, mechanical, or other
coupling mechanisms.
[0065] Various types of liquid handler units (100) are commercially
available, for example in certain aspects, a liquid handler may be
a robotic handler such as a Hamilton MICROLAB.RTM. STAR work
station or a Beckman Coulter BIOMEK.RTM. 2000 liquid handler (B2K).
See also, U.S. Pat. No. 6,325,114 concerning robotic liquid
handlers. In still other aspects, a liquid handler maybe a device
that does not comprise a robotic arm but rather moves liquid by
actuation of valves and the application of pressure gradients, such
as a fluidic or microfluidic liquid handler.
[0066] A wide array of incubators (104) are known in the art and
may be used according to embodiments of the invention. For example,
in certain embodiments an incubator may be a Kendro CYTOMAT.TM.
incubator.
[0067] Furthermore, pluripotent or ES cell expansion apparatus and
systems in certain embodiments of the invention may comprise a
controller (106) for the control of ES cell expansion. Such a
program may be in electronic communication with liquid handler unit
(100), a fluid communication device (108) and/or an incubator
(104). The skilled artisan will recognize that in certain aspects,
an operating apparatus or system may be comprised in a computer or
a computer-readable medium. An example operating program for use in
embodiments of the invention may comprise the steps depicted in
FIG. 1. In this exemplary embodiment, the operating controller
directs ES cell separation, that is effected by: (i) removing
media; (ii) contacting cells of the ES cell population with a
proteolytic chemical or enzyme such as trypsin; (iii) incubating
and agitating the cells to ensure disassociation of the cells; and
(iv) seeding the separated ES cells in fresh media. In certain
embodiments, the above-described process can be repeated to produce
additional ES cells.
[0068] As will be appreciated, the operating apparatus may be
effected by means of computer automation, whereby the operating
apparatus directs and controls the various hardware devices that
make up certain embodiments of the present invention. An exemplary
operating program that may be employed to effect integration of
hardware elements is the OVERLORD.TM. Integration software program
(Biosero, Inc.), which employs a simple drag-and-drop system for
setting up communication between instruments. The software also
permits a range of programming elements such as numeric and string
variables, conditional statements (e.g., IF THEN, ELSE), and
control loops (e.g., FORNEXT).
[0069] Optionally, an apparatus according to the invention may
comprise fluid communication device (108) that facilitates fluid
communication between incubator (104) and liquid handler unit
(100). For example, in the case where a liquid handler is a robotic
handler, fluid communication device (108) may be a robotic device,
such as a device that moves plates of cells between a liquid
handler unit and an incubator. For example, a robotic device may be
a Hudson Platecrane XL.
[0070] Furthermore, a pluripotent or ES cell expansion system may
comprise one or more reservoirs (110, 112, 114) that comprise
reagent for the liquid handler unit (100). For example, reservoirs
may comprise: cell growth media (e.g., media comprising a ROCK
inhibitor) with or without a proteinase inhibitor; cell culture
plates; a proteolytic enzyme solution; phosphate buffered saline
(PBS); and/or pipette tips. In certain aspects, additional robotic
devices may be used to facilitate communication between a liquid
handler device and a reservoir. In certain embodiments a reservoir
may contain a TeSR media, optionally with a ROCK inhibitor and/or a
protease inhibitor such as a soybean trypsin inhibitor. In other
embodiments, the reservoir may contain a solution comprising a
proteolytic enzyme (e.g., trypsin, EDTA, etc.), For example, in
some aspects a Beckman Coulter Stacker Carousel may be used to
facilitate communication between a reservoir (e.g., a plate or
pipette reservoir) and a liquid handler device. The reservoirs may
be housed in a temperature control unit, such as a refrigerator.
The temperature control unit may optionally comprise a heating unit
to pre-heat solutions to a desired temperature (e.g., about
37.degree. C.); however, the inventors have discovered that a
heating unit is not necessary in certain embodiments, as a simple
refrigerator has been successfully used in the below examples.
[0071] Referring now to FIG. 2B, a top view of an apparatus 50 for
providing automated cell culture comprises a stacker carousel
(141), a liquid handler unit (100), an incubator (104), a fluid
communication device (108), a controller (106), and a series of
reservoirs (110, 112, 114). In certain embodiments, the stacker
carousel (141) is mechanically coupled to or comprises part of the
liquid handler unit (100). As used herein, the term "reservoir"
includes any device capable of retaining a volume of fluid. It is
also understood that various components shown in FIG. 2B can be
combined or separated. For example, reservoirs (110, 112, 114) can
be integral with liquid handler unit (100), or separate from liquid
handler unit (100). In specific embodiments, fluid communication
device (108) is a robotic arm, e.g. a Hudson Platecrane XT. In
certain embodiments, liquid handler unit (100) is a Biomek2000 and
incubator 400 is a Cytomat6000 model.
[0072] In the illustrated embodiment, liquid handler unit (100)
further comprises a tool station 121 that comprises various sizes
of liquid handling tools, e.g. pipetting tools that can be used to
pipette different volumes of liquid. Tool station (121) can also
comprise a gripper tool that can be used, e.g. to remove and/or
install lids from cell culture plates during various steps of the
cell culture process. In the specific embodiment shown, liquid
handler unit (100) comprises a station (122) that includes P250
barrier tips, which can be used with MP200 pipette tools of station
(121). In addition, liquid handler unit (100) comprises a station
(123) for source plates, a station (124) for lids for daughter
plates, and a station (125) for daughter plates.
[0073] In the embodiment shown in FIG. 2B, liquid handler unit
(100) also comprises a station (132) that serves as a proteolytic
enzyme (e.g., trypsin solution) or chemical reservoir, a station
(133) that provides lids for source plates, a station (134) that
provides lids for daughter plates, and a station (135) that
provides daughter plates.
[0074] In certain embodiments, automated passaging may be
accomplished using the following exemplary protocol: After
retrieval of a mother plate from incubator (104) via fluid
communication device (108), a wash tool from station (121) removes
the spent media. A pitetting tool (e.g., an 8-channel 2000
pipetting tool MP200) from station (121) can then add about 3 ml of
trypsin (0.1%) from station (132). Fluid communication device can
then transfer the plate to incubator 104. After an incubation of
about 7 minutes, fluid communication device (108) transfers the
treated plate back to liquid handler unit (100). A mixture of 3 ml
TeSR medium containing 2 .mu.M H-1152 and 1 mg/ml Invitrogen
Soybean Trypsin Inhibitor is then added to each well from one or
more of reservoirs (110, 112, 114). The cells can then be washed
off the plate surface and mixed using a pipetting tool from station
121 by repetitive dispensing and aspiration. The cells can then be
dispensed to daughter plates provided from station 125 or 135 using
the pipetting tool from station 121.
[0075] The cells can then be seeded at a ratio of, e.g., 1:32 onto
precoated Matrigel plates loaded from the stacker carousel (131) to
the liquid handler (100) (e.g., Biomek2000). Seeding can be done
after aspirating the Matrigel coating media and replacing it with a
modified TeSR media containing H-1152 and soybean inhibitor (e.g.,
TeSR containing 1 .mu.M HA-1152 and 0.5 mg/ml Invitrogen Soybean
Trypsin Inhibitor) provided from one or more of reservoirs (110,
112, 114). Controller (106) may be used to control the movements of
liquid handler unit (100), fluid communication device (108), and/or
incubator (104). A gripper tool from station 121 may also be used
to remove or install lids from plates during appropriate steps in
the automated cell culture method.
[0076] As stated above, the H-1152 could be replaced with another
ROCK inhibitor such as H-100 if desired. In this way, the cells may
be separated and split without the need for physically of removing
the proteolytic enzyme from the growth media; for example, using
this approach, the inactivated trypsin does not need to be
physically removed from the media, e.g., via centrifugation.
[0077] In various embodiments, multiple robotic components may be
utilized to further expedite the culturing protocol and increase
the high throughput of the system. For example, multiple robotic
arms may be utilized for a operating separate tools, and a liquid
handling system like the Tecan Cellerity system, which has been
successfully established for maintenance of other attached cell
lines, may also be used with the present invention.
EXAMPLES
[0078] The following examples are included to further illustrate
various aspects of the invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
that follow represent techniques and/or compositions discovered by
the inventor to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
Example 1
Automated Passage and Expansion of Stem Cells
[0079] H1 cells passage 185 and 62 were cultured using TeSR media
and split using a Beckman Coulter Biomek 2000 liquid handler (B2K),
Gibco TrypLE Trypsin, TeSR media and TeSR Plus (containing 10 .mu.M
HA-100 and 0.5 mg/ml Invitrogen Soybean Trypsin Inhibitor). H1
cells, approximately 70% confluent, were placed on the B2K work
surface along with 6-well plates coated with 8.6 .mu.g/cm.sup.2
MATRIGEL.TM. (BD Bioscience) and a reservoir containing TRYPLE.TM.
Trypsin. Using the Gripper tool, the lid from plate to be split was
removed. The robot discarded the Gripper tool, and loaded the Washl
tool to aspirate media from the plate with cells. Next, using the
P1000 tool, TRYPLE.TM. enzyme was transferred to the plate to be
split. The lid was replaced on the plate and the plate was moved
into the 37.degree. C. CYTOMAT.TM. incubator for 7 minutes to allow
for cells to dissociate from plate.
[0080] During this time the lids from the MATRIGEL.TM. coated
plate(s) were removed and excess MATRIGEL.TM. was removed using the
Washl tool. The appropriate volume of TeSR Plus media was dispensed
per well for each MATRIGEL.TM. coated plate. The plate from the
incubator was then removed and uncovered using the Gripper tool.
The Washl tool was used to add TeSR Plus media to the plate in
order to neutralize the TRYPLE.TM. in the trypsinized well.
Remaining clumps of cells were mixed slowly and broken up using the
P1000 tool. The cell suspension was transferred to the new
MATRIGEL.TM. coated plate(s), while intermittently mixing and
slowly distributing (seeding) cells across the wells of each plate.
This process was continued until all new plates were seeded with
cells. Using the Gripper tool the lids were then placed back on
plate(s), and plate(s) were placed at 37.degree. C. degrees for 24
hours.
[0081] After 24 hours seeded plates were removed from incubator and
using the Washl tool, TeSR plus media was aspirated and fresh
regular TeSR media was added to each plate of cells. The lids were
replaced and the plates were again placed back in incubator, and
fed every 24 hours with regular TeSR media (TeSR without HA-100 and
Soybean Trypsin Inhibitor) until they needed to be split again,
approximately 4-5 days later. The method enabled cells to be
expanded from one plate to 30 plates (i.e., to a 30.times. greater
surface area) in a single split.
Example 2
Automated HES Cell Culture and Maintenance System
[0082] In order to improve labor and time-intensive maintenance of
HES cells the inventors first automated feeding of HES cells. The
proposed experiment was to maintain 10 E-well plates between
passages by automated media exchange to establish sterile and
reproducible conditions. The inventors achieved that goal by
successfully automating media change using an established liquid
handling system. This was an important step toward the automation
of HES cell culture in this embodiment.
[0083] FIGS. 3A-B show the automated system used in the below
experiments of exemplary embodiments. The system includes a
Biomek2000 System (pin tools, wash tools, stacker, single and
8-channel 20, 200 and 1000 microliter pipetting tools (P20, P200,
P1000, Beckman), a Hudson Platecrane XT, and a Heraeus Cytomat
6000. In this embodiment, a simple soda refrigerator was used for
media storage from which the media was delivered directly to the
culture wells. An in-line heating of the delivered media did not
appear to be necessary based on experiments. The integration was
done in collaboration with Biosero using Overlord software. The
complete system was housed in a class100 clean room (i.e. less than
100 particles larger than 0.5 microns per cubic feet) compliant
with biosaftety level 2 (BSL2, regulates handling of agents with
moderate potential hazard to personnel and the environment)
regulations and accomplished sufficient sterility comparable to a
standard BSL2 tissue culture hood.
[0084] Rectangular 4-well and 8-well NunclonA, plates purchased
from Nunc were used instead of the round 6-well plates commonly
used in manual procedures. One reason for this was that the plate
height of all commercially available 6-well plates significantly
exceeds those of regular microwell plates used in screening and
automated liquid handling. The penalty of using regular round
6-well plates would have been a reduction of almost half of the
capacity of the automated incubator to less than 100 plates.
Furthermore, the rectangular geometry of the plates allowed the use
of the 8-channel liquid handling tools since 6-well plates contain
more inaccessible areas. The later fact also resulted in a
1.46-fold increase in usable culture surface area (84 cm2 for 4-
and 8-well plates compared to 57.6 cm2 for a 6-well plate). The
feeding and seeding system used 4-well and 8-well plates with the
wash tool and the 8-channel tool (FIGS. 3 C-D).
[0085] In this embodiment, robotic components were used for
splitting and feeding HES cells. The single channel wash tool was
used initially to aspirate spent media and feed 4-well or Swell
plates. In the back behind the 4-well plate a 6-well plate is used
to serve as a reservoir for trypsin. The 8-channel tool P200 was
used to mix and dispense the trypsinized and individualized HES
cells from a 4-well mother plate to an 8-well daughter plate in the
final split.
[0086] In exemplary embodiments, the surface was NunclonA,
manufactured by Nunc and coated with Matrigel. The following
robotic components can be used for splitting and feeding HES cells:
the Platecrane connects the liquid handling robot Biomek2000 with
the incubator Cytomat6000. The gripper of the Platecrane is
positioned above the turntable of the Incubator. The refrigerator
housing the media had tubing connecting the peristaltic pump of the
wash tool (box with the tape) and the waste bottle for spent media
coming from the vacuum was controlled by a valve in the wash tool
which was provided by an in house vacuum system.
[0087] Initially, Omnitrays, i.e. plates with no divisions within
the whole plate, were tested but were abandoned because of
excessive splashing during transfers by the Platecrane and the
turntable of the Cytomat6000. In the final feeding protocol
(initially handled 6-well plates) two 8-well plates were retrieved
from the Cytomat6000 one at a time and transferred to the two most
outer right positions of the Biomek2000 deck using the Platecrane.
The gripper tool of the Biomek2000 removed the lids from the plates
and placed them on the adjacent left positions on the deck. After
switching to the 8-channel wash tool the media is aspirated at 8
positions across the 4- or 8-well plate to insure sufficient
removal of spent media. In the manual process the plate would be
tipped at an angle to collect the spent media at the bottom for
sufficient removal. Such an angle was not implemented with these
robotics to maintain reliability. The new media (6 and 3 ml per
well respectively) was dispensed immediately before moving on to
the next well or plate. The delidding and relidding process as well
as the height alignment of the wash tool required occasional
intervention. These were the most prominent causes (about 1 in 100
movements) of errors that required operator presence and
intervention. The flow rate of the wash tool did not seem to
negatively affect the quality of the culture as the cells could not
be washed off the surface by the pressure generated by the
peristaltic pump of the wash tool. The quality of the culture was
judged by visual assessment of the culture after each day of the
culturing process (FIGS. 4A-C).
[0088] The automated procedure did not show any increased
occurrence of differentiation during the 5 days of the experiment
and the cultures could be successfully split manually onto regular
Matrigel plates. Initial experiments with an in-line heating column
to preheat the chilled media were not continued as the inventors
did not observe any negative effect without the preheating column.
The major and time-consuming task in this aim was to master the
automated system for the key movements and operations that were
similar during feeding and splitting. The throughput of this system
for the step of feeding was somewhat limited due to: limitations
including in the number of simultaneous movements possible by the
integration software, the Biomek2000 being used to operate multiple
tools (in the case of feeding: gripper and wash tool), and the
configuration with the Platecrane and the available deck space of
the Biomek2000 allowing simultaneous processing of two plates.
However, a larger deck offered by more advanced liquid handling
systems and a more sophisticated integration platform (multiple
arms, compatible software, integratable robotics that allow
multitasking) significant improvements in throughput can and have
to be made. By the end of this study including aim 2 the inventors
were able to demonstrate successfully that the inventors can
maintain 160 NunclonA, 8-well plates between passages by automated
media exchange to establish sterile and reproducible conditions. A
single person can routinely handle about 20 NunclonA, 6-well plates
per day without compromising culture quality. The above system
clearly demonstrated good culture quality, measurable in
maintenance of pluripotency (Oct4 levels), speed, reproducibility
and economic efficiency of the HES cell culture comparable with
manually maintained cell culturing techniques.
Example 3
Automated Passaging and Expansion of hES Cells
[0089] Since passaging of HES cells is the most labor intensive and
variable step in HES cell culture it leads to tremendous
variability in the outcome of experiments depending highly on the
skills of the technician. The inventors hypothesized that
automation of passaging will lead to more robust and reproducible
HES cell culture. Although the inventors could not confirm this
theory with the current system and due to the limited time
available for this project, the inventors were able to demonstrate
a proof of principle by expanding one well of a 6-well plate of HES
cells (-2.5 million cells) to a final number of 160 plates
(.about.2-3 billion cells) equivalent to a 1000-fold expansion over
3 weeks, an otherwise nearly impossible task for a single
person.
[0090] The inventors utilized the system described in Example 1 and
developed procedures for passaging of HES cells that were based on
simple liquid handling protocols. Recent innovations in splitting
techniques allowed for efficient automation of this otherwise
demanding manual procedure. Since HES cells require cell-cell
contacts for survival in TeSR media, they needed to be seeded in
clumps which required scraping of attached cells, a procedure which
would be very hard to automate. However, the small molecule HA-100
and its 10-fold more specific derivative H-1152 was determined to
allow the survival of HES cells after trypsin treatment. The
ability to detach and individualize HES cells with 0.1% trypsin and
subsequent seeding onto Matrigel coated NunclonA, plates in
slightly modified defined TeSR medium containing 1 .mu.M H-1152 and
0.5 mg/ml Invitrogen Soybean Trypsin Inhibitor allowed the
inventors to adopt techniques that have been automated by others
for less demanding adherent cancer cell lines.
[0091] For automated passaging, the following procedure was
developed: After retrieval of the mother plate from the incubator,
the wash tool removes the spent media as described in aim 1. The
8-channel 2000 pipetting tool MP200 is used to add 3 ml of trypsin
(0.1%). After 7 minute incubation of the treated plate inside the
Cytomat6000, a mixture of 3 ml TeSR medium containing 2 .mu.M
H-1152 and 1 mg/ml Invitrogen Soybean Trypsin Inhibitor is added to
the well. The cells are washed off the plate surface and mixed
using the 8-channel MP200 tool by repetitive dispensing and
aspiration. Cells are then dispensed to the daughter plates using
the MP200 tool. The cells are then seeded 1 to 32 onto precoated
Matrigel plates loaded from the stacker to the Biomek2000. Seeding
is done after aspirating the Matrigel coating media and replacing
it with the modified TeSR media containing H-1152 and soybean
inhibitor as described in the feeding protocol above. The total
surface area of the initially used Omnitray turned out to be too
large for the throughput of the system at the stage of the next
split once the inventors tested methods for passaging HES cells in
aim 2. The time it took to distribute the cells from the mother
plates to an average of 25 daughter plates exceeded the time the
individualized cells could survive in suspension before they would
be seeded.
[0092] The inventors used karyotypically normal very high passage
(>p200) H1 HES cell cultures in the beginning to establish the
robotic protocols with very robust growing cells to establish
feasibility of these new protocols. These cells still produced
hematopoietic precursors and cardiomyocytes to demonstrate their
differentiation potential. However the demands for a robust
protocol are certainly greater for lower passage cell cultures,
since they react more sensitively to sub-optimal conditions, which
leads to greater variability in cell culture. In later experiments,
the inventors were able to confirm the validity of the derived
procedures with lower passage H1 cells (>p60) in smaller scale
experiments with 5 plates over 3 to 5 passages. This evidence
supports the ability to culture these lower passage cells using the
automated system.
[0093] Initially the inventors used the single channel tool for
seeding by adding cells at multiple positions of the well to
achieve homogeneous distribution of cells. Although the inventors
could accomplish that successfully with high and low passage HES
cultures, the throughput of the procedure required the use of the
8-channel P200 tool when the inventors expanded the cells in the
final step of the scale-up experiment to 160 8-well plates.
Although a reduced homogeneous distribution and decrease in cell
densities of the cells was observed in the daughter plates, the
inventors anticipate that this method may be optimized to improve
these characteristics. The expansion in this experiment was done
from a single well of a 6-well plate to 5 needed 4-well plates in
the first passage and then into 160 final 8-well plates in the
second passage. The inventors maintained all 160 plates by feeding
2 days after passage with the protocol established above and then
every day. The inventors inspected all plates visually for
differentiation and density. The inventors randomly picked 30
plates and stained them with trypan blue staining for scanning and
evaluation of cell distribution. From the visual inspection of the
plates it was apparent that further improvements can and should be
made to homogeneity in the interest of high reproducibility. Also
the inventors picked plate 2 and 10 randomly to test for Oct4
content of the cells after 2 passages and 1000-fold expansion over
20 days. Oct4 FACS analysis revealed a high quality of
undifferentiated cells in this first scale-up experiment.
[0094] No significant amount of differentiation was observed in any
plate achieving a standard quality of automated HES cells culture.
This was supported by many other experiments in smaller scale and
with lower passage cultures as well as by the very high content of
Oct4 positive cells in the FACS analysis. The passaging frequency
and density to the current cell culture depended on its history and
predominantly age (i.e. passage number). From experience with
manual HES cell culture the inventors have learned that younger
cultures have the tendency to react more sensitive to suboptimal
conditions and harsh treatment than older cells. The behavior of a
cell line can vary depending on how it is thawed and handled in the
first few passages. All these factors can contribute to a large
degree of variability that can only be harnessed by monitoring the
growth and degree of differentiation. Such measures may be
automated in subsequent variations of the system described here. As
shown in FIG. 6, variability was observed amongst the plates.
Improved mixing procedures and integrated cell-counting may
accommodate better homogeneity in the future systems, these
modifications should be relatively easily implemented since they
have been accomplished with other cell cultures before.
[0095] Randomly picked plates from the scale-up experiment after
1000-fold expansion were examined. Plates were stained with trypan
blue after media had been removed and the plates were dried. The
dye marked the HES cell colonies. Although there remains a need for
improved cell distribution when optimizing the splitting protocol,
the inventors anticipate that improved consistency in the
distribution across the plates can be achieved with optimization of
the protocol.
[0096] Randomly picked plates from the scale-up experiment after
1000-fold expansion were subjected to cell counts. Plates were
trypsinized and stained with trypan blue and counted on a
hemacytometer. Total cells were calculated from representative
samples. Extrapolated from the mean results shown in the below
summary statistics, approximately 160.times.16 million=2.56 billion
cells were generated from the above experiments. Cell expansion in
different plates are shown in FIG. 6, and the cell expansion
summary statistics for this experiment are presented in Table 2
below.
TABLE-US-00002 TABLE 2 Cell Expansion Results Summary Statistics
Mean 16023750 Standard Error 2615974.1 Median 13000000 Mode
16500000 Standard Deviation 12815603.45 Sample Variance 1.6424E+14
Kurtosis 0.987042208 Skewness 1.254713927 Range 44970000 Minimum
3000000 Maximum 47970000 Sum 384570000 Count 24
Example 4
Automated Seeding of HES Cell Culture for 96-Well Format for
Cell-Based Screening
[0097] The inventors used a new compound H1152 which actively
increased colony formation of HES cells in TeSR1 media from
individualized cells and represents a 2nd generation small molecule
derived from HA-100. HA-100 was discovered in one of the first HES
cell-based small molecule screens which led to the discovery of
related compounds like H1152 in follow-up studies. H-1152 allows
for very efficient seeding of individualized HES cells in 96-well
plates (similar to HA-100 but at 10-fold lower concentration),
enabling HES cell-based small molecule screening. Individualized
HES cells that are otherwise passaged in cell clumps allow more
uniform cell densities per well, which is a stringent prerequisite
for cell-based small molecule screening. The inventors also
reasoned that the current small molecule (HA-100) and a related
compound H1152 were sufficient for passaging human ES cells.
[0098] The inventors used a small molecule library (2000 compounds
with known bioactivity) to screen for compounds that may have the
ability to increase hematopoiesis. The inventors have demonstrated
that the system described above is capable of plating HES cells in
96-well format to provide a platform for small molecule screening
as well as screening of other agents and conditions. The inventors
have derived a screening assay and protocol suitable for automation
that uses directed differentiation methods leading to hematopoietic
precursers. The inventors have successfully performed a screen
under these conditions and identified 28 initial candidate
compounds which are now being validated. Although the performance
of the screen can be further improved through a more thorough assay
development, the key goal of plating HES cells for automated
screening has been accomplished. This platform allows for the
production of large quantities of these cells that are otherwise
difficult to obtain in quantities required for studies involved in
screens, toxicology testing and target validation. The alternative
source (cells isolated from blood and bone marrow) offers only
limited quantities and can not provide a stable genetic background,
due to the fact that batches from different donors have to be
pooled. Using these methods, cells with reproducible quality may be
provided to the research community.
[0099] The assay used in this protocol was developed based on an
ELISA protocol which can detect the presence of two characteristic
cell surface markers (CD34 and CD43). These markers identify potent
hematopoietic precursors that the inventors can subsequently
isolate to differentiate the cells into the desired blood lineages
or provide as such as a starting material. The inventors used the
automated platform to seed individualized human ES cells in 96-well
plates and grow them for four days. Then the media was switched to
defined differentiation media and compound to be screened was added
by the robot to a final concentration of 20 micromolar using a
96-channel pin tool. After four days exposure to the compound
including an addition of media after two days the media was changed
to a growth factor reduced differentiation media and maintained for
6 more days (with media changes every other day) until the cells
are subjected to the ELISA protocol for final readout. The timeline
of this experiment was developed for differentiation media only.
The inventors have obtained 1-6% CD43 and 2-25% CD34 positive cells
by this method. The screen was designed to detect compounds that
further increase the population of potent hematopoietic precursors
to increase production. Due to issues with the CD34 antibody, data
for only CD43 expression was obtained. Nonetheless, the population
of CD43 positive cells is likely the most important marker to
identify a commitment to the blood lineages.
[0100] The inventors were able to use the AmplexUltraRed and the
SensiFlex assays (Invitrogen) in a multiplex ELISA and did show
with controls, that these assays were compatible and offer the
possibility for SCP to simultaneously screen for two cell markers
(on the surface or inside the cells, after addition of a separate
wash step in the protocol). This is particularly important when
screening for cell lineages that require more than one marker for
sufficient recognition. A high rate of false positives amongst
control DMSO wells at the edge of some plates as well as an
accumulation of hits among the top and bottom row of some plates.
Although this last study conducted yielded preliminary data, the
inventors this screening assay may be optimized at the stage of the
ELISA, through better washing procedures and with optimized
antibodies and concentrations, as has been demonstrated in other
ELISA screens. Nonetheless, successfully providing HES cells for
screening in 96-well plates represents a key accomplishment.
[0101] In this application the inventors were able to obtain very
good homogeneity in plating into 96-well plates using a modified
procedure established above. For this purpose however the inventors
plated 16,000 cells per well, which is a 5-fold higher seeding
density, than in the regular propagation and maintenance of
undifferentiated HES cells with the automated procedure. This
particular change was used in order to accommodate attachment to an
alternate proprietary matrix other than Matrigel, which turned out
to be a crucial step in the differentiation protocol. Successful
seeding in 96-well format to facilitate robust screening may be
used.
[0102] Although the inventors had the capacity to perform the media
changes for this screen with the automated liquid handling system
at hand, the inventors decided in the interest of time and money,
to change media and perform the ELISA assay, by aspirating manually
using a vacuum 12-channel wand and dispensing using an automated
dispenser without stacker. While the inventors did not take
advantage of the reproducibility of a fully automated system, the
inventors saved significant time and reagents. A Matrix Wellmate
automated dispenser was used.
[0103] The above examples demonstrate a successfully automated HES
cell culture and maintenance. The above data demonstrates that the
inventors were able to solve the major challenges towards providing
an automatable procedure for HES cell culture. The inventors
further anticipate that additional throughtput can be achieved via
optimization of the system. The inventors anticipate that improved
quality control, monitoring of cell growth, and improved passaging
can be achieved using the above system. It is anticipated that with
an optimized procedure, one may not need to identify and isolate
differentiated impurities as is necessary in manual protocols.
[0104] The above system may be easily modified to include a liquid
handling system like the Tecan Cellerity system, which has been
successfully established for maintenance of other attached cell
lines. The major step towards the application of such a system was
the use of trypsin to passage cells. An independent publication by
Watanabe et al. Nat Biotech (2007) "A ROCK inhibitor permits
survival of dissociated human embryonic stem cells" supports the
ability of HA-100 to maintain HES cell growth.
[0105] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
REFERENCES
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