U.S. patent application number 15/789464 was filed with the patent office on 2018-03-08 for cancer cell enrichment system.
The applicant listed for this patent is XCELL BIOSCIENCES, INC.. Invention is credited to Luke CASSEREAU, James LIM.
Application Number | 20180066223 15/789464 |
Document ID | / |
Family ID | 61282016 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180066223 |
Kind Code |
A1 |
LIM; James ; et al. |
March 8, 2018 |
CANCER CELL ENRICHMENT SYSTEM
Abstract
Embodiments of the invention relate to a cell culture incubator
having a gas flow regulation system that exerts control over
atmospheric parameters within the incubator. Particular embodiments
include an enclosed environmental chamber and a control unit
operably linked thereto, the control unit having an oxygen module
and a pressure module. Control unit embodiments, by way of these
modules, are configured to regulate both oxygen partial pressure
and total gas pressure within the enclosed environmental chamber.
Embodiments of the control unit are adapted (a) to provide
instructions to the oxygen module to regulate an oxygen level to an
instructed hypoxic oxygen level and (b) to provide instructions to
the pressure module to regulate total gas pressure to an instructed
positive pressure level. The regulation of oxygen to the instructed
hypoxic level prevails despite an oxygen partial
pressure-increasing effect of the positive pressure condition
associated with the instructed positive pressure level.
Inventors: |
LIM; James; (Oakland,
CA) ; CASSEREAU; Luke; (Emeryville, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
XCELL BIOSCIENCES, INC. |
San Francisco |
CA |
US |
|
|
Family ID: |
61282016 |
Appl. No.: |
15/789464 |
Filed: |
October 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15566337 |
Oct 13, 2017 |
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PCT/US2016/027881 |
Apr 15, 2016 |
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15789464 |
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62149268 |
Apr 17, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/48 20130101;
C12M 41/34 20130101; C12M 41/40 20130101; C12M 41/14 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/34 20060101 C12M001/34; C12M 1/36 20060101
C12M001/36 |
Claims
1-26. (canceled)
27. A cell culture incubator comprising: an enclosed environmental
chamber; and a control unit operably linked to the enclosed
environmental chamber, wherein the control unit comprises an oxygen
module and a pressure module, wherein the control unit is
configured to regulate each of an oxygen level and a total gas
pressure within the enclosed environmental chamber, and wherein the
control unit is adapted to: a) provide instructions to the oxygen
module to regulate the oxygen level to an instructed hypoxic oxygen
level; and b) provide instructions to the pressure module to
regulate the total gas pressure to an instructed positive pressure
level, wherein the regulation of the oxygen level to the instructed
hypoxic level prevails despite an oxygen partial pressure
increasing effect of a positive pressure condition, per the
instructed positive pressure level.
28. The cell culture incubator of claim 27, further comprising: an
oxygen sensor configured to measure the oxygen level within the
enclosed environmental chamber and to convey an informative signal
to the oxygen module; and a pressure sensor configured to measure
the total atmospheric gas pressure within the enclosed
environmental chamber and to convey an informative signal to the
pressure module.
29. The cell culture incubator of claim 27, further comprising a
nitrogen source operably connected to the enclosed environmental
chamber, wherein a flow of nitrogen is regulated by the control
unit.
30. The cell culture incubator of claim 29, wherein the regulated
nitrogen flow is directed into the enclosed environmental chamber
by way of a chamber gas flow path, and wherein the regulated
nitrogen flow comprises a response to oxygen sensor data via the
oxygen module, wherein in the response to a sensed oxygen level
that is above the instructed oxygen level, the response comprises
an instruction to flow nitrogen into the environmental chamber.
31. The cell culture incubator of claim 30, wherein as a result of
a dilution of oxygen within the enclosed environmental chamber by
the flow of nitrogen, the sensed oxygen level reaches the
instructed oxygen level, and wherein the control unit then
instructs a cessation of the nitrogen flow into the environmental
chamber.
32. The cell culture incubator of claim 29, wherein the regulated
nitrogen flow is directed into the enclosed environmental chamber
by way of a chamber gas flow path, and wherein the regulated
nitrogen flow comprises a response to pressure sensor data by way
of the pressure module, wherein the response to a pressure level
that is below the instructed pressure level comprises an
instruction to flow nitrogen into the environmental chamber.
33. The cell culture incubator of claim 32, wherein as a result of
an increase in pressure level within the enclosed environmental
chamber by the flow of nitrogen, the pressure level comes reaches
the instructed pressure level, and wherein the control unit then
instructs a cessation of the nitrogen flow into the environmental
chamber.
34. The cell culture incubator of claim 28, wherein the regulation
of pressure within the enclosed environmental chamber by the
control unit comprises a response to the pressure sensor, as
mediated by the pressure module.
35. The cell culture incubator of claim 34, wherein the regulation
of pressure within the enclosed environmental chamber comprises a
response to a pressure lower than the instructed pressure level,
and wherein the response to the high pressure comprises an inflow
of nitrogen.
36. The cell culture incubator of claim 34, wherein the regulation
of pressure within the enclosed environmental chamber comprises a
response to a pressure lower than the instructed pressure level,
and wherein the enclosed environmental chamber further comprises a
carbon dioxide source operably connected to the enclosed
environmental chamber, wherein a flow of carbon dioxide is
regulated by the control unit, and wherein the response to the high
pressure comprises an inflow of carbon dioxide.
37. The cell culture incubator of claim 36, wherein the regulation
of pressure within the enclosed environmental chamber comprises a
response to a pressure lower than the instructed pressure level,
and wherein the response to the low pressure comprises a cessation
of inflow of carbon dioxide or inflow of nitrogen.
38. The cell culture incubator of claim 36, wherein the regulation
of carbon dioxide flow into the enclosed environmental chamber by
the control unit comprises a response to a carbon dioxide sensor
configured to measure the carbon dioxide level within the enclosed
environmental chamber.
39. The cell culture incubator of claim 36, wherein the regulation
of carbon dioxide flow into the enclosed environmental chamber by
the control unit comprises a response to the pressure sensor, as
mediated by the pressure module.
40. The cell culture incubator of claim 27, wherein the oxygen
level within the enclosed environmental chamber is regulated by the
oxygen module, and wherein the oxygen module provides instructions
to regulate any one or both of a flow of nitrogen or a flow of
carbon dioxide into the enclosed environmental chamber.
41. The cell culture incubator of claim 27, wherein the
instructions to regulate to an instructed hypoxic level comprise
instructions to adjust the oxygen level to a value within a range
of about 0.1% to about 21% oxygen.
42. The cell culture incubator of claim 27, wherein the
instructions to regulate to an instructed hypoxic level comprise
instructions to adjust the oxygen level to a value within a range
of about 1.0% to about 12% oxygen.
43. The cell culture incubator of claim 27, wherein the
instructions to regulate to an instructed hypoxic level comprise
instructions to adjust the oxygen level to a value within a range
of about 2% to about 6% oxygen.
44. The cell culture incubator of claim 27, wherein the pressure
level within the enclosed environmental chamber is regulated by the
pressure module, wherein the pressure module provides instructions
to regulate any one or more of a flow of nitrogen or a flow of
carbon dioxide.
45. The cell culture incubator of claim 27, wherein the
instructions to regulate pressure to an instructed positive
pressure level comprise instructions to adjust the pressure to a
value within a range of about 0.5 PSIG to about 30 PSIG.
46. The cell culture incubator of claim 27, wherein the
instructions to regulate pressure to an instructed positive
pressure level comprise instructions to adjust the pressure to a
value within a range of about 1.0 PSIG to about 20 PSIG.
47. The cell culture incubator of claim 27, wherein the
instructions to regulate pressure to an instructed positive
pressure level comprise instructions to adjust the pressure to a
value within a range of about 2.0 PSIG to about 10 PSIG.
48. The cell culture incubator of claim 27, wherein the
instructions to regulate pressure to an instructed positive
pressure level comprise instructions to adjust the pressure to a
value within a range of about 2.5 PSIG to about 5.0 PSIG.
Description
CROSS REFERENCE
[0001] This Application is a continuation-in-part of U.S.
application Ser. No. 15/566,337, filed Oct. 13, 2017, which is a
national stage entry of PCT/US2016/027881, filed Apr. 15, 2016,
which claims the benefit of U.S. Provisional Application No.
62/149,268, filed Apr. 17, 2015, each of which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to instruments and systems for cell
culture. More particularly, the invention relates to cell culture
systems that can regulate aspects of the atmospheric environment
within a cell culture incubator chamber.
BACKGROUND
[0003] Cell enrichment systems can be used to enrich, isolate, and
expand different populations of cells. These cell populations can
include, for example, cancer cells, circulating tumor cells, stem
cells, and immune cells. Isolation and characterization of
different cell types that are induced through a cell enrichment
system can be used to understand tumor etiology, the biology of
metastasis, stem cell differentiation, immune cell proliferation,
and to provide a biomarker for tumor progression.
[0004] Atmospheric conditions, such as the atmospheric pressure and
the concentrations of particular gases, such as oxygen, can be
significant factors in cell culture that can affect the growth
rate, viability, and expression of phenotypic aspects of various
cell populations. To understand these biological effects, cell
culture instruments that can regulate various atmospheric
conditions precisely and independently of each other could be
valuable for research, diagnostic, and therapeutic goals.
INCORPORATION BY REFERENCE
[0005] Each patent, publication, and non-patent literature cited in
the application is hereby incorporated by reference in its entirety
as if each was incorporated by reference individually.
SUMMARY OF THE INVENTION
[0006] Embodiments of the invention relate to a cell culture
incubator that includes gas flow regulation system that exerts
control over the atmospheric parameters to which cells in culture
are exposed. Particular embodiments of the invention include an
enclosed environmental chamber and a control unit operably linked
to the enclosed environmental chamber, the control unit having an
oxygen module and a pressure module. Embodiments of the control
unit, by way of these modules, are configured to regulate both
oxygen level and total gas pressure within the enclosed
environmental chamber. Embodiments of the control unit are adapted
(a) to provide instructions to the oxygen module to regulate an
oxygen level to an instructed hypoxic oxygen level and (b) to
provide instructions to the pressure module to regulate total gas
pressure to an instructed positive pressure level. Embodiments may
also be instructed to operate at an ambient oxygen level and an
ambient pressure level. The oxygen level and the total gas pressure
level are regulated independently of each other. The regulation of
oxygen to the instructed hypoxic level prevails despite the oxygen
partial pressure-increasing effect of the positive pressure
condition associated with the instructed positive pressure
level.
[0007] Some embodiments of the cell culture incubator further
include one or more oxygen sensors configured to measure the oxygen
level within the enclosed environmental chamber and to convey an
informative signal to the oxygen module, and one or more pressure
sensors configured to measure the total atmospheric gas pressure
within the enclosed environmental chamber and to convey an
informative signal to the pressure module. Both the oxygen module
and the pressure module are within the control unit. The control
unit may include further control units related to the regulation of
atmospheric parameters. Typically, atmospheric control units are
configured to receive sensory input from the enclosed environmental
chamber or the ambient environment, and to direct instructions to
other elements of a gas flow regulation system.
[0008] Embodiments of a cell culture incubator and an included gas
flow regulation system may include a nitrogen source operably
connected to the cell culture incubator, wherein the flow of
nitrogen is regulated by the control unit.
[0009] In some of these gas flow regulation embodiments, the
regulated nitrogen flow is directed into the enclosed environmental
chamber by way of a chamber gas flow path, wherein the regulated
nitrogen flow includes a response to oxygen sensor data by way of
the oxygen module, and wherein the response to a sensed oxygen
level that is above the instructed oxygen level includes an
instruction to flow nitrogen into the environmental chamber. In
such embodiments and consequent response, as a result of a dilution
of oxygen within the enclosed environmental chamber by the inflow
of nitrogen, the sensed oxygen level may come into compliance with
the instructed oxygen level. At that point, the control unit may
then instruct a cessation of the nitrogen flow into the
environmental chamber.
[0010] In some of these gas flow regulation system embodiments for
a cell culture incubator, the regulated nitrogen flow is directed
into the enclosed environmental chamber by way of a chamber gas
flow path, the regulated nitrogen flow includes a response to
pressure sensor data by way of the pressure module, wherein the
response to a pressure level that is below the instructed pressure
level includes an instruction to flow nitrogen into the
environmental chamber. In such embodiments and consequent response,
as a result of an increase in pressure level within the enclosed
environmental chamber brought about by the inflow of nitrogen, the
pressure level may come into compliance with the instructed
pressure level. At that point, the control unit may then instruct a
cessation of the nitrogen flow into the environmental chamber.
[0011] In some of these gas flow regulation system embodiments for
a cell culture incubator, the regulation of pressure within the
enclosed environmental chamber by the control unit includes a
response to the pressure sensor, as mediated by the pressure
module. In some of these particular embodiments, the regulation of
pressure within the enclosed environmental chamber includes a
response to a pressure lower than the instructed pressure level,
wherein such response to the high pressure includes an inflow of
nitrogen. In some embodiments, the regulation of pressure within
the enclosed environmental chamber includes a response to a
pressure lower than the instructed pressure level, wherein such
response to the high pressure includes an inflow of carbon dioxide.
In some embodiments, the regulation of pressure within the enclosed
environmental chamber includes a response to a pressure lower than
the instructed pressure level, wherein such response to the low
pressure includes a cessation of inflow of carbon dioxide or a
cessation of inflow of nitrogen.
[0012] Some of these gas flow regulation system embodiments for a
cell culture incubator include regulation of the level of carbon
dioxide, at least in part, to engage a pH buffering system within
the cell culture medium. In some of these embodiments, the
regulation of carbon dioxide flow into the enclosed environmental
chamber by the control unit include a response to the carbon
dioxide sensor, as mediated by the carbon dioxide module. In some
of these embodiments, the regulation of carbon dioxide flow into
the enclosed environmental chamber by the control unit includes a
response to the pressure sensor, as mediated by the pressure
module.
[0013] Particular embodiments of these gas flow regulation system
embodiments for a cell culture incubator are directed toward
regulating oxygen level within the incubator to a hypoxic level.
Accordingly, the oxygen level within the enclosed environmental
chamber is regulated by the oxygen module, the oxygen module
providing instructions to regulate any one or both of a flow of
nitrogen or a flow of carbon dioxide into the enclosed
environmental chamber. Inasmuch as oxygen level is typically
regulated to a level lower than that of the ambient level,
approaches to lowering oxygen include dilution by addition of
nitrogen or carbon dioxide, with a venting in order to keep total
gas pressure at an instructed level. In the event that the oxygen
level drifts below the instructed level, input of air by an air
injection pump provides an oxygen source.
[0014] In some embodiments of a gas flow regulation system, the
instructions to regulate to an instructed hypoxic level include
instructions to adjust the oxygen level to a value within a range
of about 0.1% to about 21% oxygen. In other embodiments, the
instructions to regulate to an instructed hypoxic level include
instructions to adjust the oxygen level to a value within a range
of about 1.0% to about 12% oxygen. In other embodiments, the
instructions to regulate to an instructed hypoxic level include
instructions to adjust the oxygen level to a value within a range
of about 2% to about 6% oxygen.
[0015] Particular embodiments of these gas flow regulation system
embodiments for a cell culture incubator are directed toward
regulating the total gas pressure within the incubator to a level
that is greater than the ambient total gas pressure. The total gas
pressure units used here (PSIG) refer to an amount of pressure over
the ambient atmospheric pressure. In typical embodiments of the gas
flow regulation system, the pressure level within the enclosed
environmental chamber is regulated by the pressure module, the
pressure module providing instructions to regulate any one or more
of a flow of nitrogen or a flow of carbon dioxide, the inflow of
either gas resulting in an increased pressure.
[0016] In some embodiments of the gas flow regulation system that
are directed to creating a high pressure condition, the
instructions to regulate pressure to an instructed positive
pressure level include instructions to adjust the pressure to a
value within a range of about 0.5 PSIG to about 30 PSIG. In some
embodiments, the instructions to regulate pressure to an instructed
positive pressure level include instructions to adjust the pressure
to a value within a range of about 1.0 PSIG to about 20 PSIG. In
some embodiments, the instructions to regulate pressure to an
instructed positive pressure level include instructions to adjust
the pressure to a value within a range of about 2.0 PSIG to about
10 PSIG. In some embodiments, the instructions to regulate pressure
to an instructed positive pressure level include instructions to
adjust the pressure to a value within a range of about 2.5 PSIG to
about 5.0 PSIG.
[0017] In some embodiments, the invention provides a cell culture
incubator, wherein the cell culture incubator comprises: a) an
enclosed environmental chamber; and b) a control unit, wherein the
control unit is operably linked to the enclosed environmental
chamber, wherein the control unit comprises a computer program
product comprising a computer-readable medium having
computer-executable code encoded therein, the computer-executable
code adapted to encode: (i) an oxygen level module, wherein the
oxygen level module is encoded to regulate an oxygen level of the
enclosed environmental chamber, wherein the oxygen level module is
encoded to control the removal of oxygen in the enclosed
environmental chamber to generate a hypoxic oxygen level within the
enclosed environmental chamber; (ii) a pressure module, wherein the
pressure module is encoded to regulate the pressure of the enclosed
environmental chamber, wherein the pressure module controls the
addition of gas to generate a positive pressure condition in the
enclosed environmental chamber; (iii) a temperature module, wherein
the temperature module is encoded to regulate the temperature of
the enclosed environmental chamber; and (iv) a humidity module,
wherein the humidity module is encoded to regulate the humidity of
the enclosed environmental chamber, wherein each of the oxygen
level, pressure, temperature, and humidity mimics an in vivo
microenvironment for a cell, wherein the cell culture incubator
reaches each of an instructed oxygen level, pressure, temperature,
and humidity within about 20 minutes of receiving an input of each
of the instructed oxygen level, pressure, temperature, and
humidity.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is an immunofluorescence image of a representative
CTC cluster.
[0019] FIG. 2 is an illustrative workflow for enrichment of a
target subpopulation of cells.
[0020] FIG. 3 depicts enrichment and propagation of a target
subpopulation of cells using a method of the invention.
[0021] FIG. 4 is an illustrative computer system to be used with a
method of the invention.
[0022] FIG. 5 depicts results of biomarker assessment in CTCs from
prostate cancer cells.
[0023] FIG. 6 shows results of biomarker determination for prostate
cancer cells.
[0024] FIG. 7 displays results of mRNA sequencing analysis for the
nerve growth factor signaling pathway.
[0025] FIG. 8 displays results of mRNA sequencing analysis for the
Aurora A signaling pathway.
[0026] FIG. 9 displays results of mRNA sequencing analysis for the
Kit receptor signaling pathway.
[0027] FIG. 10 is an immunofluorescence image for EPCAM expression
in a PDAC CTC.
[0028] FIG. 11 depicts results of NANOG signaling pathway
expression in PDAC versus mCRPC CTCs.
[0029] FIG. 12 depicts results of Wnt signaling pathway expression
in PDAC versus mCRPC CTCs.
[0030] FIG. 13 displays results of SNP and INDEL analysis for
CTCs.
[0031] FIG. 14 displays results of SNP and INDEL analysis for
CTCs.
[0032] FIG. 15 depicts an illustrative user interface for a system
of the invention.
[0033] FIG. 16 depicts an illustrative user interface for a system
of the invention.
[0034] FIG. 17 depicts an illustrative user interface for a system
of the invention.
[0035] FIG. 18 depicts an illustrative user interface for a system
of the invention.
[0036] FIG. 19 displays an illustrative cell culture incubator of
the invention.
[0037] FIG. 20 displays a door configuration of a cell culture
incubator of the invention.
[0038] FIG. 21 displays a door configuration of a cell culture
incubator of the invention.
[0039] FIG. 22 depicts a door heater of a cell culture incubator of
the invention.
[0040] FIG. 23 depicts increased cellular transfection efficiency
using green fluorescent protein.
[0041] FIG. 24A is an illustrative embodiment of gas flow control
system to be used with a cell culture incubator and a method of the
invention.
[0042] FIG. 24B is an illustrative embodiment of nitrogen flow
system to be used with a cell culture incubator and a method of the
invention.
DETAILED DESCRIPTION
Method of the Invention
[0043] A method of the present invention can be used to isolate
CTCs, and other target cell subpopulations, from a biological
sample. A method of the invention can be used, for example, to
isolate cell populations, selectively separate cell populations,
maintain cells in a differentiated or an undifferentiated state,
forcibly differentiate cells, enrich cell populations, expand cell
populations (through proliferation or selective enrichment),
modulate functions of cell populations, modulate morphology of cell
populations, modulate epigenetic characteristics, and modulate gene
and protein expression profiles. A method of the invention can be
used in, for example, primary cells, cell lines, or microbial
communities.
[0044] Target cell subpopulations can include, for example, CTCs,
cancer stem cells (CSCs), hematopoietic stem cells (HSCs),
endothelial progenitor cells (EPCs), pre-cancerous cells, stem
cells, fetal stem cells, undifferentiated stem cells, fetal cells,
bone marrow cells, progenitor cells, foam cells, mesenchymal cells,
epithelial cells, epithelial progenitor cells, endothelial cells,
endometrial cells, trophoblasts, cancer cells, red blood cells,
white blood cells, immune system cells, connective tissue cells,
hepatocytes, neurons, induced pluripotent stem (IPS) cells, or any
combination thereof.
[0045] A method of the invention can be used, for example, to
maintain neuronal cells in culture, to maintain hepatocytes in
culture, and toxicity screening. The invention can be used to
differentiate IPS cells and stems cell into, for example, cells of
the mesoderm, ectoderm, and endoderm. A method of the invention can
be used to differentiate cells into neurons, cardiomyocytes,
hepatocytes, hematopoietic stem cells, osteoblasts, osteoclasts,
epithelial cells, endothelial cells, astrocytes, adipocytes, immune
cells, mast cells, erythrocytes, oocytes, or spermatocytes.
[0046] CTCs can be composed of heterogeneous clusters of cancer and
immune cells in vivo and can display differential expression of
immunomodulatory and stem cell signaling pathway in vitro.
[0047] FIG. 1 displays immunofluorescence (IF) for a circulating
tumor cell cluster from a subject with castration-resistant
prostate cancer (CRPC). The IF image demonstrates that CTCs can
contain both cancer and immune cells. The DAPI staining (indicated
by oval-shaped cell staining) indicates the nuclei of the cells. A
white blood cell (WBC) antibody cocktail can be used to detect, for
example, CD3, CD14, CD16, CD19, CD20, CD45, and CD56, in the CTC
cluster (white arrows in FIG. 1). A cytokeratin antibody cocktail
can be used to detect, for example, CK4, CK5, CK6, CK8, CK10, CK13,
and CK18 in the CTC cluster (fibrillar staining seen in and around
oval-shaped nuclei in FIG. 1). The bright white staining, and the
fibrillar staining around the oval-shaped nuclei, indicates
prostate-specific membrane antigen (PSMA) and prostate-specific
antigen (PSA) proteins. The numbers in the legend indicate the
wavelength (nm) used for excitation of the stains.
[0048] FIG. 2 depicts an illustrative workflow that can be used to
obtain an enriched population of cells from a heterogeneous cell
population. The sources of cells used in a method of the invention
can include, for example, fresh blood, white blood cells (WBC), the
buffy layer of centrifuged blood, cryopreserved tumors and
biopsies, samples for leukapheresis, fine needle aspirates, fresh
biopsies, urine, or fecal matter. After obtaining a sample from a
subject, the sample can be prepared for use in a cell isolation kit
to separate, for example, a blood sample into plasma, white blood
cells and platelets, and red blood cells. CTCs can be found in the
white blood cell and platelet fraction of centrifuged blood. The
cell isolation step can be about 30 minutes long. After the
heterogeneous cell population has been isolated, the cells can be
applied to the enrichment medium for propagation and enrichment of
viable CTC colonies. The adhesion of the cells to the substrate can
take from a few hours to about one day. Proliferation during
culture of the cells can be observed within about 1 to 3 days after
adherence, after which next-generation sequencing (NGS) can be
performed on the cell colonies for the markers of interest.
Next-generation sequencing methods can include, for example, whole
genome sequencing, whole genome resequencing, whole exome
sequencing, whole transcriptome mRNA sequencing, ChIP-sequencing,
and bioinformatics.
[0049] FIG. 3 depicts adherence and propagation of a collected
heterogeneous cell population from a subject. First, the cells can
be applied to a plate, which can be coated with a substance, such
as a growth factor-infused hydrogel. After about 30 minutes, the
cells are adhered to the plate. Over the next two hours, the cells
spread across the plate, and the growth medium is exchanged
allowing for removal of any remaining white bloods cells (WBCs).
The media is replaced with a chemically-defined culture medium to
promote the growth of CTC colonies. The cells can be grown in an
environment that can be adjusted to mimic, for example, the tumor
microenvironment, via changes in oxygen or pressure levels to
obtain viable CTCs. In some embodiments, the cells are grown in
hypoxic conditions.
[0050] The present invention can use a substrate to capture target
cell subpopulations from a sample. The heterogeneous cell
population can be applied to, for example, a culture dish coated
with a substrate that can promote growth and enrichment of the
target cell population. The target cell subpopulation can adhere to
the substrate with higher affinity than other cells, for example,
white blood cells. Cells that do not adhere to the substrate can be
washed away with media or maintained in culture. Once adhered, the
cells can spread and begin dividing on the substrate.
[0051] The substrate can comprise, for example, 1, 2, 3, 4, or 5
layers. The distance between two substrates layers may range from
about 0.001 to about 20 mm, about 1 to about 10 mm, or about 1 to
about 5 mm and each layer can be about 0.001, about 1, about 2,
about 3, about 4, about 5, about 6, about 7, about 8, about 9,
about 10, about 12, about 15, about 17, or about 20 mm.
[0052] The cells can be plated on a material made of, for example,
plastic, glass, gelatin, polyacrylamide, or any combination
thereof. The dishes used to the plate the cells can be, for
example, microscope slides, culture plates, culture dishes, Petri
dishes, microscope coverslips, an enclosed environmental chamber, a
sealed culture dish, or multi-well culture dishes.
[0053] The binding surface layer of the substrate can be the
portion of the substrate that is in contact with the captured
cells. In some instances, the binding surface layer is the only
layer, adjacent to the base layer, or separated from the base layer
by one or more middle layers.
[0054] The binding surface layer of the substrate can comprise, for
example, cell monolayers, cell lysates, biological materials
associated with the extracellular matrix (ECM), gelatin, or any
combination thereof.
[0055] Biological materials associated with the ECM can include,
for example, collagen type I, collagen type IV, laminin,
fibronectin, elastin, reticulin, vimentin, hygroscopic molecules,
glycosaminoglycanse, proteoglycans, roteoglycans, glycocalyx,
bovine serum albumin, human serum albumin, Poly-L-lysine,
Poly-D-lysine, or Poly-L-ornithine. The gelatin can be from an
animal source, for example, the gelatin can porcine or bovine.
[0056] The monolayer of cells used in the substrate can be, for
example, mammalian cells, endothelial cells, vascular cells, venous
cells, capillary cells, human umbilical vein endothelial cells
(HUVEC), human lung microvascular endothelial cells (HLMVEC), human
keratinocytes, human mesenchymal stem cells, human bone marrow
stromal cells, and human astroglial cells. The cell lines can be
obtained from a primary source or from an immortalized cell line.
The monolayer of cells can be irradiated by ultraviolet light or
X-ray sources to cause senescence of cells. The monolayer can also
contain a mixture of one or more different cell types. The
different cell types may be co-cultured together. One non-limiting
example of co-culture is a combination of primary human endothelial
cells co-cultured with transgenic mouse embryonic fibroblasts mixed
to form a monolayer.
[0057] The binding surface layer of the substrate can contain, for
example, a mixture of intracellular components. One method that can
be used to obtain a mixture of intracellular components is lysis of
the cells and collection of the cytosolic and cytoskeletal
components. The lysed cells may be primary or immortalized. The
lysed cells can be from either mono- or co-cultures.
[0058] The binding surface layer of the substrate can contain
biological materials associated with the extracellular matrix (ECM)
or binding moieties such as hyaluronic acid hydrogels. For example,
gelatin can be mixed directly with cells, binding moieties,
biological materials associated with the ECM, or any combination
thereof, to make a binding surface layer for the substrate. For
example, the binding surface layer can comprise a gelatin mixed
with a collagen.
[0059] The substrate can have one or more middle layers. The middle
layer of the substrate can be one or more monolayers of cells. The
cells of the monolayer can be of varying origin. For example, the
middle layer of the substrate can be made by growing a confluent
monolayer of mouse embryonic fibroblasts on the base layer and then
growing another layer of cells, for example, the binding surface
layer, on top of the confluent mouse embryonic fibroblasts.
[0060] A feeder layer can be used in the substrate for growth and
enrichment of the target cell subpopulation. A feeder layer can sit
adjacent to a base layer and can be separated from the binding
surface layer of the substrate. The feeder layer can be a monolayer
of feeder cells. The cells of the monolayer can be of varying
origin. For example, the feeder layer can be made by growing a
monolayer of human endothelial cells or mouse embryonic fibroblasts
on a base layer.
[0061] Conjugation of layers of the substrate can be done by
allowing cells to grow in a monolayer on top of the base layer or
middle layer. Conjugation of layers can also be done by
pre-treating the surface with a surface of either net positive, net
negative, or net neutral charge. The conjugation procedure can be
aided by chemical moieties, linkers, protein fragments, nucleotide
fragments, or any combination thereof.
[0062] The configuration and composition of the substrate can be
tailored for enrichment of a particular target cell subpopulation.
The composition of the substrate can vary based on, for example,
patient type, cancer type, stage of cancer, patient medical
history, and genomic and proteomic analysis of the patient
tumor.
[0063] The enrichment media used for growing the cells can be
supplemented or made with culture media that has been collected
from cell cultures, blood plasma, or any combination thereof. The
enrichment media can be, for example, Plating Culture Medium, Type
R Long Term Growth Medium, Type DF Long Term Growth Medium, Type D
Long Term Growth Medium, and MEF--Enrichment Medium, or any
combination thereof. The enrichment medium can contain, for
example, a primary nutrient source, animal serum, ions, elements,
calcium, glutamate, magnesium, zinc, iron, potassium, sodium, amino
acids, vitamins, glucose, growth factors, hormones, tissue
extracts, proteins, small molecules, or any combination
thereof.
[0064] Non-limiting examples of amino acids that can used in the
enrichment media include essential amino acids, phenylalanine,
valine, threonine, tryptophan, isoleucine, methionine, leucine,
lysine, and histidine, arginine, cysteine, glycine, glutamine,
proline, serine, tyrosine, alanine, asparagine, aspartic acid,
glutamic acid, or any combination thereof.
[0065] Non-limiting examples of growth factors that can be used in
the enrichment media include epidermal growth factor (EGF), nerve
growth factor (NGF), brain derived neurotrophic factor (BDNF),
fibroblast growth factor (FGF), stem cell factor (SCF),
insulin-like growth factor (IGF), transforming growth factor-beta
(TGF-.beta.), basic fibroblast growth factor (bFGF), testosterone,
estrogen, thyroid-stimulating hormone (TSH), follicle-stimulating
hormone, luteinizing hormone, eicosanoids, melatonin, thyroxine,
vasopressin, oxytocin, or any combination thereof.
[0066] Non-limiting examples of hormones include peptide hormones,
insulin, steroidal hormones, hydrocortisone, progesterone,
testosterone, estrogen, dihydrotestosterone, or any combination
thereof.
[0067] Non-limiting examples of tissue extracts include pituitary
extract. Non-limiting examples of small molecule additives include
sodium pyruvate, endothelin-1, transferrin, cholesterol, or any
combination thereof.
[0068] Non-limiting examples of other components that can be used
in the enrichment media include pipecolic acid, gamma-Aminobutyric
acid (GABA), human serum albumin, bovine serum albumin,
glutathione, human alpha-fetoprotein, bovine alpha-fetoprotein,
human holo-transferrin, or any combination thereof.
[0069] Non-limiting examples of salts that can be used in the
enrichment media include calcium chloride, magnesium chloride,
sodium bicarbonate, magnesium sulfate, sodium chloride, citrate,
potassium phosphate, sodium phosphate, or any combination
thereof.
[0070] In some embodiments, the enrichment media contains pipecolic
acid, GABA, bFGF, TGF.beta.-1, human insulin, human
holo-transferrin, human serum albumin, and reduced glutathione.
[0071] The amino acids, growth factors, hormones, tissue extracts,
salts, or any other component that can be used in the enrichment
media can be at a concentration of, for example, about 0.001 nM,
about 0.005 nM, about 0.01 nM, about 0.015 nM, about 0.02 nM, about
0.25 nM, about 0.03 nM, about 0.035 nM, about 0.04 nM, about 0.045
nM, about 0.05 nM, about 0.055 nM, about 0.06 nM, about 0.065 nM,
about 0.07 nM, about 0.075 nM, about 0.08 nM, about 0.085 nM, about
0.09 nM, about 0.1 nM, about 0.015 nM, about 0.2 nM, about 0.25 nM,
about 0.3 nM, about 0.35 nM, about 0.4 nM, about 0.45 nM, about 0.5
nM, about 0.55 nM, about 0.6 nM, about 0.65 nM, about 0.7 nM, about
0.75 nM, about 0.8 nM, about 0.85 nM, about 0.9 nM, about 0.95 nM,
about 0.001 .mu.M, about 0.005 .mu.M, about 0.01 .mu.M, about 0.015
.mu.M, about 0.02 .mu.M, about 0.025 .mu.M, about 0.03 .mu.M, about
0.035 .mu.M, about 0.04 .mu.M, about 0.045 .mu.M, about 0.05 .mu.M,
about 0.055 .mu.M, about 0.06 .mu.M, about 0.065 .mu.M, about 0.07
.mu.M, about 0.075 .mu.M, about 0.08 .mu.M, about 0.085 .mu.M,
about 0.09 .mu.M, about 0.085 .mu.M, about 0.09 .mu.M, about 0.085
.mu.M, about 0.09 .mu.M, about 0.085 .mu.M, about 0.09 .mu.M, about
0.085 .mu.M, about 0.09 .mu.M, about 0.085 .mu.M, about 0.09 .mu.M,
about 0.085 .mu.M, about 0.09 .mu.M, about 0.095 .mu.M, about 0.1
.mu.M, about 0.15 .mu.M, about 0.2 .mu.M, about 0.25 .mu.M, about
0.3 .mu.M, about 0.35 .mu.M, about 0.4 .mu.M, about 0.45 .mu.M,
about 0.5 .mu.M, about 0.55 .mu.M, about 0.6 .mu.M, about 0.65
.mu.M, about 0.7 .mu.M, about 0.75 .mu.M, about 0.8 .mu.M, about
0.85 .mu.M, about 0.9 .mu.M, about 0.95 .mu.M, about 0.001 mM,
about 0.005 nM, about 0.01 mM, about 0.015 mM, about 0.02 mM, about
0.025 mM, about 0.03 mM, about 0.035 mM, about 0.04 mM, about 0.045
mM, about 0.05 mM, about 0.055 mM, about 0.06 mM, about 0.065 mM,
about 0.07 mM, about 0.075 mM, about 0.08 mM, about 0.085 mM, about
0.09 mM, about 0.095 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM,
about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about
0.45 mM, about 0.5 mM, about 0.55 mM, about 0.6 mM, about 0.65 mM,
about 0.7 mM, about 0.75 mM, about 0.8 mM, about 0.85 mM, about 0.9
mM, about 0.95 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM,
about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about
10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35
mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70
mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120
mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about
170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM,
about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500
mM, about 600 mM, about 700 mM, about 800 nM, about 900 mM, and
about 1 M.
[0072] The culturing conditions in a method of the invention can be
adjusted to simulate oxygen and pressure levels found in a
particular microenvironment to promote the collection of a desired
cell population. The microenvironment can be, for example, a tumor
microenvironment, bone metastatic environment, vasculature
environment, or brain microenvironment. The oxygen level used
during culturing conditions or in a cell incubator can be, for
example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about
0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%,
about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about
8%, about 9%, about 10%, about 11%, about 12%, about 13%, about
14%, about 15%, about 16%, about 17%, about 18%, about 19%, about
20%, about 21%, about 22%, about 23%, about 24%, or about 25%
oxygen in the incubator. In some embodiments, the cells can be
grown under hypoxic conditions.
[0073] The culturing condition in a method of the invention can be
adjusted to simulate the pressure found in the tumor
microenvironment to promote the collection of, for example, CTCs,
maintenance of a tumor biopsy, or expansion of a tumor biopsy. The
pressure used during culturing conditions can be a PSI gauge (PSIG)
reading of, for example, about 0.5 PSIG, about 0.6 PSIG, about 0.7
PSIG, about 0.8 PSIG, about 0.9 PSIG, about 1 PSIG, about 1.1 PSIG,
about 1.2 PSIG, about 1.3 PSIG, about 1.4 PSIG, about 1.5 PSIG,
about 1.6 PSIG, about 1.7 PSIG, about 1.8 PSIG, about 1.9 PSIG,
about 2 PSIG, about 2.5 PSIG, about 3 PSIG, about 3.5 PSIG, about 4
PSIG, about 4.5 PSIG, about 5 PSIG, about 6 PSIG, about 7 PSIG,
about 8 PSIG, about 9 PSIG, about 10 PSIG, about 15 PSIG, about 20
PSIG, about 25 PSIG, about 30 PSIG, about 35 PSIG, about 40 PSIG,
about 45 PSIG, about 50 PSIG, or about 55 PSIG.
[0074] The pressure used during culturing conditions can be, for
example, about 3.45 kPa, about 4.14 kPa, about 4.83 kPa, about 5.52
kPa, about 6.21 kPa, about 6.89 kPa, about 7.58 kPa, about 8.27
kPa, about 8.96 kPa, about 9.65 kPa, about 10.3 kPa, about 11 kPa,
about 11.7 kPa, about 12.4 kPa, about 13.1 kPa, about 13.8 kPa,
about 17.2 kPa, about 20.7 kPa, about 24.1 kPa, about 27.6 kPa,
about 31 kPa, about 34.4 kPa, about 41.4 kPa, about 48.3 kPa, about
55.2 kPa, about 62.1 kPa, about 68.9 kPa, about 103 kPa, about 138
kPa, about 172 kPa, about 207 kPa, about 241 kPa, about 276 kPa,
about 310 kPa, about 345 kPa, or about 379 kPa.
[0075] The pH of the enrichment media used in a method of the
invention can be, for example, about 2, about 2.1, about 2.2, about
2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about
2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about
3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about
4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.55, about
4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.5, about 6,
about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7,
about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,
about 7.7, about 7.8, about 7.9, about 8, about 8.5, about 9, about
9.5, about 10, about 10.5, or about 11 pH units.
[0076] The viscosity of the enrichment media can be adjusted by,
for example, at least 0.001 Pascal-second (Pas), at least 0.001
Pas, at least 0.0009 Pas, at least 0.0008 Pas, at least 0.0007 Pas,
at least 0.0006 Pas, at least 0.0005 Pas, at least 0.0004 Pas, at
least 0.0003 Pas, at least 0.0002 Pas, at least 0.0001 Pas, at
least 0.00005 Pas, or at least 0.00001 Pas, depending on the cell
types being cultured.
[0077] The oxygen solubility of the enrichment media can be, for
example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about
0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%,
about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about
8%, about 9%, about 10%, about 11%, about 12%, about 13%, about
14%, about 15%, about 16%, about 17%, about 18%, about 19%, about
20%, about 21%, about 22%, about 23%, about 24%, about 25%, about
26%, about 27%, about 28%, about 29%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
96%, about 97%, about 98%, or about 99%.
[0078] A method of the invention can further comprise coating
surfaces for cell adhesion with particular media compositions to
promote cellular and cellular protein binding to the surface. The
surface can be, for example, a cell culture plate, a cell culture
plate with multiple wells, a petri dish, a glass slide, a cover
slip, or a glass dish. The media used for coating of the cell
adhesion surfaces can include, for example,
3-(aminopropyl)-trimethoxysilane,
(3-mercapto-propyl)trimethoxysilane,
(3-Aminopropyl)triethoxysilane, N-[3
-(trimethoxysilyl)-propyl]-ethylenediamine,
(3-Glycidyloxypropyl)trimethoxysilane,
[3-(2-aminoethyl-amino)-propyl]trimethoxysilane,
trimethoxy[3-(methylamino)propyl]silane,
3-aminopropyl(diethoxy)-methylsilane, or glutaraldehyde.
[0079] The surface coating can further comprise an extracellular
matrix (ECM) mix to facilitate cell binding. The mix can include,
for example, collagens, basement membrane proteins, collagen IV,
laminins, fibronectin, vitronectin, vimentin, tumor-derived
extracellular matrix proteins, or inert self-assembling peptides
systems. The components used can be animal- or human-derived. The
ECM mix can be diluted to a pH of about 4 to about 10 using, for
example, potassium hydroxide (KOH), 1-glycine, DMEM powder, sodium
hydroxide (NaOH), or PBS. The ECM mix can be further supplemented
with, for example, human plasma or animal-derived serum. The
animal-derived serum can be, for example, bovine serum or fetal
bovine serum.
[0080] The cells can be cultured in enrichment media, or in a cell
culture incubator, for about 1 minute, about 2 minutes, about 3
minutes, about 4 minutes, about 5 minutes, about 10 minutes, about
15 minutes, about 20 minutes, about 25 minutes, about 30 minutes,
about 40 minutes, about 50 minutes, about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 10 hours, about 11
hours, about 12 hours, about 18 hours, about 24 hours, about 30
hours, about 36 hours, about 42 hours, about 2 days, about 3 days,
about 4 days, about 5 days, about 6 days, about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, about 1 year, about 1.5 years, about 2 years, about 2.5
years, or about 3 years.
[0081] Databases containing information regarding genetic mutations
that are prevalent in specific types of cancer can be used to
compare the genetic profile or biomarker expression of the target
subpopulations derived using the present invention to known
mutations. Non-limiting examples of databases that can be used for
comparison include COSMIC, cBio Portal, Human Gene Mutation
Database (HGMD TM), GWAS central, and the Universal Mutation
Database.
Cell Culture Incubator
[0082] The invention further provides a cell culture incubator. The
cell culture incubator can comprise an enclosed environmental
chamber. The cell culture incubator can be configured to maintain a
gas composition of the enclosed environmental chamber, an
atmospheric pressure of the enclosed environmental chamber,
humidity, carbon dioxide level, oxygen level, and an internal
ambient temperature of the enclosed environmental chamber. The cell
culture incubator can comprise a control unit, wherein the control
unit can be configured to maintain at least one of the gas
composition, the atmospheric pressure, humidity, carbon dioxide
level, oxygen level, or the internal ambient temperature. The
control unit can be operably linked to the enclosed environmental
chamber. The control unit can be configured to maintain at least
two of the gas compositions, the atmospheric pressure, humidity,
carbon dioxide level, oxygen level, and the internal ambient
temperature. The control unit can be configured to maintain the gas
composition, the atmospheric pressure, humidity, carbon dioxide
level, oxygen level, and the internal ambient temperature. The
control unit of the cell culture incubator can be user-controlled
or automated based on sensors in the cell culture incubator. The
control unit can be configured to create a dynamic gas composition,
atmospheric pressure, humidity, carbon dioxide level, oxygen level
and the internal ambient temperature as a function of time. The
control unit can be configured to cycle between several different
gas compositions, atmospheric pressure, humidity level, carbon
dioxide level, oxygen level, and the internal ambient temperature
as a function of time, and can be stochastic or periodic. Some
aspects of embodiments of the cell culture incubator are described
further below in the section entitled "Gas Flow Regulation System",
and are depicted in FIGS. 24A-24B.
[0083] At least one of the gas composition, the atmospheric
pressure, humidity, carbon dioxide level, oxygen level, and the
internal ambient temperature can be configured for selective
proliferation of a target primary cell subpopulation as compared to
a non-target primary cell subpopulation. The selective
proliferation of a target cell subpopulation can be evidenced by,
for example, a two-fold increase in the proliferation rate of the
target primary cell subpopulation as compared to the proliferation
rate of the non-target primary cell subpopulation. At least one of
the gas composition, the atmospheric pressure, humidity, carbon
dioxide level, oxygen level, and the internal ambient temperature
can be configured for selective adherence of a target primary cell
subpopulation as compared to a non-target primary cell
subpopulation. The selective adherence of a target primary cell
subpopulation can be evidenced by a two-fold increase in adherence
of the target primary cell subpopulation as compared to adherence
of the non-target primary cell subpopulation. At least one of the
gas composition, the atmospheric pressure, humidity, carbon dioxide
level, oxygen level, and the internal ambient temperature can be
configured to promote selective colony formation of the target
primary cell as compared to colony formation of the non-target
primary cell subpopulation. The selective colony formation can be
evidenced by a two-fold increase in colony formation of the target
primary cell subpopulation as compared to colony formation of the
non-target primary cell subpopulation. The colony formation can be
a two-dimensional or three-dimensional colony formation.
[0084] The gas composition in the cell culture incubate can
comprise an oxygen level between about 0.1 to about 21%. In some
embodiments, the cell culture incubator maintains an oxygen level
of the enclosed environmental chamber of no more than about 5%. In
some embodiments, the cell culture incubator maintains an oxygen
level of the enclosed environmental chamber of no more than about
2%. In some embodiments, the cell culture incubator maintains an
oxygen level of the enclosed environmental chamber of no more than
about 1%.
[0085] The cell culture incubator can maintain a user-controlled or
automated atmospheric pressure of the enclosed environmental
chamber of about 1 PSIG (6.89 kPa) or greater. In some embodiments,
the cell culture incubator maintains a user-controlled atmospheric
pressure of the enclosed environmental chamber of about 2 PSIG
(13.8 kPa) or greater. In some embodiments, the cell culture
incubator maintains a user-controlled atmospheric pressure of the
enclosed environmental chamber of about 5 PSIG (34.5 kPa). The cell
culture incubator can maintain the atmospheric pressure of the
enclosed environmental chamber by controlling an inlet gas
pressure.
[0086] The cell culture incubator can comprise a user interface.
The user interface can be configured to allow a user to control the
gas composition, oxygen level, carbon dioxide level, humidity,
atmospheric pressure, or internal ambient temperature. The user
interface can be configured to provide a display of the gas
composition to a user. The user interface can be configured to
provide a display of the oxygen level, carbon dioxide level,
humidity, atmospheric pressure of the enclosed environmental
chamber, and internal ambient temperature to a user. The cell
culture incubator can be configured to maintain an internal
humidity of the enclosed environmental chamber. The enclosed
environmental chamber can comprise a shelf, a pressure sensor, an
oxygen sensor, a carbon dioxide sensor, a temperature sensor, or an
oxygen removal catalyst. The shelf in the enclosed environmental
chamber can be made of, for example, stainless steel, silver, gold,
or copper. In some embodiments, the shelf of the enclosed
environmental chamber is a copper shelf.
[0087] The cell culture incubator can be operably linked to a gas
tank. The gas tank can comprise a CO2 tank, a nitrogen gas tank, an
oxygen gas tank, a gas tank comprising a defined mixture of one or
more gases, or any combination thereof. The cell culture incubator
can be operably linked to the gas tank via a pressurized pump or
pressure sensor (e.g., a pressure gauge). The pressurized pump or
pressure sensor can maintain a controlled flow of gas from the gas
tank to the enclosed environmental chamber of the cell culture
incubator. The controlled flow of gas from the one or more tanks
can have a set inlet pressure, the set inlet pressure of the one or
more tanks configured to maintain the desired internal gas
composition or internal atmospheric pressure of the enclosed
environmental chamber. The enclosed environmental chamber can have
a vacuum seal on the door of the enclosed environmental chamber.
The enclosed environment chamber can be sealed by an inflatable
seal.
[0088] The incubator can comprise a pressurized door. In some
embodiments, the incubator comprises an outer pressurized door and
an inner pressurized door. The outer pressurized door and/or inner
pressurized door can be, e.g., a double-walled door. The
double-walled door can have a vacuum-sealed latch. The outer
pressurized door may include an integrated pressure sensor on the
door. The incubator can comprise a door entry. The door entry can
provide an entrance into an enclosed environmental chamber.
Dimensions of the door entry opening can be less than the
pressurized door. The door entry can comprise a rubber gasket. The
rubber gasket can create a pressurized seal.
[0089] The cell culture incubator can comprise an integrated
pressure sensor. The integrated pressure sensor can be a manifold
pressure sensor. The integrated pressure sensor can be a
water-based or silicon based pressure sensor. The incubator can
comprise a sterilization unit. The sterilization unit can be a
UV-based sterilization unit. The UV-based sterilization unit can be
configured to provide UV rays to the entire space of an enclosed
environmental chamber of the incubator. The incubator can comprise
a CO2 sensor. The CO2 sensor can be configured to provide a
detectable alarm upon deviation of +/-0.5% from a defined CO2 level
of the enclosed environmental chamber. The incubator can comprise
an enclosed environmental chamber. The incubator can comprise a
water humidity tray. The water humidity tray can promote sterility
of the enclosed environmental chamber. The water humidity tray may
be tethered directly to humidity sensor or regulator. The incubator
can comprise an air jacket. The air jacket can maintain optimal
temperature and proper gas regulation. The air jacket can be a
physically distinct compartment. The air jacket may house
electrical controllers and circuit boards. The incubator can
comprise an oxygen removal catalyst and sensor for regulating
oxygen levels within the incubator.
[0090] The incubator can comprise a heating element or temperature
control. The heating element or temperature control can comprise
silent fan-based heating elements. The silent fan-based heating
elements can dispense a constant flow of heated air into the
air-jacket compartment. Passive heating can then warm the inner
chamber in an evenly distributed and constant manner. The incubator
can comprise a pressurized pump and regulator configured to provide
a defined gas composition and internal atmospheric pressure. A
motor based pump can dispense defined gas mixtures to maintain
chamber pressure and gas composition levels (e.g., 1% oxygen, 5%
CO2, 94% N2). The incubator can comprise a user interface. A user
can use the user interface to set gas levels and pressures. The
user interface can be integrated to the sensor and pump for direct
control. The pressurized pump and regulator can comprise a gas
inlet. The gas inlet can allow flow of any gas into the enclosed
environmental chamber. For example, the gas inlet can be connected
to an oxygen tank. The gas inlet can be connected to a CO2 tank.
The gas inlet can be connected to a nitrogen tank. In some
embodiments, the incubator comprises a gas inlet connected to an
oxygen tank, a gas inlet connected to a CO2 tank, and a gas inlet
connected to a nitrogen tank. The gas inlet can be connected to a
tank containing a custom gas mixture. In some embodiments, a user
can control a flow of gas through any gas inlet. Any one of the gas
inlets may be connected to a flow meter. The flow meter can
regulate an inlet gas pressure. The cell culture incubator can
comprise a humidity control unit/sensor. The humidity control
unit/sensor can be directly connected to the water humidity
tray.
[0091] The enclosed environmental chamber of the cell culture
incubator can comprise a sterilization unit, which can be a UV
sterilization unit. The enclosed environmental chamber can comprise
a pressurized door. The enclosed environmental chamber can comprise
a sensor that provides a detectable alarm upon detection of an
oxygen level of the enclosed environmental chamber that differs by
more than about .+-.0.5% from a user-desired oxygen level. The
enclosed environmental chamber can comprise a sensor that provides
a detectable alarm upon detection of an atmospheric pressure of the
enclosed environmental chamber that differs by more than about
.+-.0.5% from a user-desired atmospheric pressure. The enclosed
environmental chamber can comprise a user display that displays an
atmospheric pressure level of the enclosed environmental chamber.
In some embodiments, the enclosed environmental chamber comprises a
user display that displays an O2 level of the enclosed
environmental chamber. In some embodiments, the enclosed
environmental chamber comprises a user display which displays a CO2
level of the enclosed environmental chamber. In some embodiments,
the enclosed environmental chamber comprises a user display which
displays a temperature level of the enclosed environmental
chamber.
[0092] A user can program the cell culture incubator to mimic, for
example, physiological, tumor microenvironment, hypoxic, high
pressure, low pressure, or supraphysiological conditions. The cell
culture incubator can be configured to calibrate to the conditions
set by the user within about one minute, about 2 minutes, about 3
minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7
minutes, about 8 minutes, about 9 minutes, about 10 minutes, about
11 minutes, about 12 minutes, about 13 minutes, about 14 minutes,
about 15 minutes, about 16 minutes, about 17 minutes, about 18
minutes, about 19 minutes, about 20 minutes, about 21 minutes,
about 22 minutes, about 23 minutes, about 24 minutes, about 25
minutes, about 30 minutes, about 35 minutes, about 40 minutes,
about 45 minutes, about 50 minutes, about 55 minutes, or about one
hour. In some embodiments, the cell culture incubator can reach the
desired conditions set by the user in less than about 20 minutes.
In some embodiments, the cell culture incubator can reach the
desired conditions set by the user in about 20 minutes. In some
embodiments, the cell culture incubator can reach the desired
conditions set by the user within 20 minutes.
[0093] In some embodiments, the enclosed environmental chamber
occupies no more than 6 cubic feet of space. In some embodiments,
the enclosed environmental chamber occupies no more than 3.5 cubic
feet of space. In some embodiments, the enclosed environmental
chamber occupies no more than 2 cubic feet of space. In some
embodiments, the enclosed environmental chamber occupies no more
than 1.5 cubic feet of space. In some embodiments, the enclosed
environmental chamber occupies no more than 1 cubic foot of space.
In some embodiments, the enclosed environmental chamber occupies
less than 1 cubic foot of space. In some embodiments, the cell
culture plate comprises 1, 6, 12, 24, 48, 96, 384, 1056, 1536, or
3456 wells.
[0094] A method of the invention can employ a cell culture
incubator for culturing of a target cell population. FIG. 19
provides an illustrative example of a cell culture incubator that
can be used in a system of the invention. 1901 is the door of the
incubator that can be opened to place a cell culture in the
incubator. 1902 is a control unit that can be used to program the
cell culture incubator using parameters including, for example,
temperature, humidity, oxygen level, carbon dioxide level, time of
incubation, nitrogen level, and chamber pressure. 1903 is a USB
port that can be used to input data to or extract data from the
cell culture incubator.
[0095] FIG. 20 is a diagram of the front of the cell culture
incubator. FIG. 20 depicts the door, a rubber gasket around the
edge of the door that can fit tightly using a door handle latch,
retaining lip, open and close buttons for the door, touch screen,
rotary actuator or motor, and chamber of the incubator. FIG. 21
shows the cell culture incubator door closed (2101) and open
(2102). FIG. 22 depicts a door heater than can be used in a system
of the invention. The figure shows the front plate, heating
element, poron insulation, back plate, mica washers, copper plate,
precision hollow shaft, and paddles that can be used to heat the
cell culture incubator to a desired temperature.
[0096] The height, width, depth, or length of the cell culture
incubator can be, for example, about 6 in, about 6.5 in, about 7
in, about 7.5 in, about 8 in, about 8.5 in, about 9 in, about 9.5
in, about 10 in, about 10.5 in, about 11 in, about 11.5 in, about
12 in, about 12.1 in, about 12.2 in, about 12.3 in, about 12.4 in,
about 12.5 in, about 12.6 in, about 12.7 in, about 12.8 in, about
12.9 in, about 13 in, about 13.1 in, about 13.2 in, about 13.3 in,
about 13.4 in, about 13.5 in, about 13.6 in, about 13.7 in, about
13.8 in, about 13.9 in, about 14 in, about 14.5 in, about 15 in,
about 15.5 in, about 16 in, about 16.5 in, about 17 in, about 17.5
in, about 18 in, about 18.5 in, about 19 in, about 19.5 in, about
20 in, about 20.5 in, about 21 in, about 21.5 in, about 22 in,
about 22.5 in, about 23 in, about 23.5 in, about 24 in, about 24.5
in, about 25 in, about 25.5 in, about 26 in, about 26.5 in, about
27 in, about 27.5 in, about 28 in, about 28.5 in, about 29 in,
about 29.5 in, about 30 in, about 30.5 in, about 31 in, about 31.5
in, about 32 in, about 32.5 in, about 33 in, about 33.5 in, about
34 in, about 34.5 in, about 35 in, about 35.5 in, about 36 in,
about 36.5 in, about 37 in, about 37.5 in, about 38 in, about 38.5
in, about 39 in, about 39.5 in, about 40 in, about 40.5 in, about
41 in, about 41.5 in, about 42 in, about 42.5 in, about 43 in,
about 43.5 in, about 44 in, about 44.5 in, about 45 in, about 45.5
in, about 46 in, about 46.5 in, about 47 in, about 47.5 in, about
48 in, about 48.5 in, about 49 in, about 49.5 in, about 50 in,
about 50.5 in, about 51 in, about 51.5 in, about 52 in, about 52.5
in, about 53 in, about 53.5 in, about 54 in, about 54.5 in, about
55 in, about 55.5 in, about 56 in, about 56.5 in, about 57 in,
about 57.5 in, about 58 in, about 58.5 in, about 59 in, about 59.5
in, about 60 in, or any combination thereof.
[0097] In some embodiments, the height of the cell culture
incubator is 12 in. In some embodiments, the width of the cell
culture incubator is 13.5 in. In some embodiments, the depth of the
cell culture incubator is 13.1 in.
[0098] The capacity of the enclosed environmental chamber can be,
for example, about 100 inch3, about 110 inch3, about 120 inch3,
about 130 inch3, about 140 inch3, about 150 inch3, about 160 inch3,
about 170 inch3, about 180 inch3, about 190 inch3, about 200 inch3,
about 205 inch3, about 210 inch3, about 211 inch3, about 212 inch3,
about 213 inch3, about 214 inch3, about 215 inch3, about 216 inch3,
about 217 inch3, about 218 inch3, about 219 inch3, about 220 inch3,
about 221 inch3, about 222 inch3, about 223 inch3, about 224 inch3,
about 225 inch3, about 226 inch3, about 227 inch3, about 228 inch3,
about 229 inch3, about 230 inch3, about 240 inch3, about 250 inch3,
about 260 inch3, about 270 inch3, about 280 inch3, about 290 inch3,
about 300 inch3, about 310 inch3, about 320 inch3, about 330 inch3,
about 340 inch3, about 340 inch3, about 350 inch3, about 360 inch3,
about 370 inch3, about 380 inch3, about 390 inch3, about 400 inch3,
about 420 inch3, about 440 inch3, about 460 inch3, about 480 inch3,
or about 500 inch3. In some embodiments, the capacity of the
enclosed environmental chamber is about 220 inch3. In some
embodiments, the capacity of the enclosed environmental chamber is
about 221 inch3. In some embodiments, the capacity of the enclosed
environmental chamber is about 222 inch3. In some embodiments, the
capacity of the enclosed environmental chamber is about 223 inch3.
In some embodiments, the capacity of the enclosed environmental
chamber is about 224 inch3. In some embodiments, the capacity of
the enclosed environmental chamber is 224 inch3.
[0099] Materials that can be used in the manufacture of the cell
culture incubator include, for example, stainless steel, glass,
copper, silver, gold, plastic, blanket batting, hard-board
insulation, or any combination thereof. The enclosed environmental
chamber can be made of, for example, copper or stainless steel. In
some embodiments, the enclosed environmental chamber is made of
copper.
[0100] The cell culture incubator can be maintained at a desired
humidity level. The humidity level can be, for example, about 70%,
about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,
about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,
about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,
about 89%, about 90%, about 90.5%, about 91%, about 91.5%, about
92%, about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%,
about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about
97.5%, about 98%, about 98.5%, about 99%, about 99.5%, or about
99.9%.
[0101] The CO2 levels in the cell culture incubator can be, for
example, about 10%, about 9.5%, about 9%, about 8.5%, about 8%,
about 7.5%, about 7%, about 6.9%, about 6.8%, about 6.7%, about
6.6%, about 6.5%, about 6.4%, about 6.3%, about 6.2%, about 6.1%,
about 6%, about 5.9%, about 5.8%, about 5.7%, about 5.6%, about
5.5%, about 5.4%, about 5.3%, about 5.2%, about 5.1%, about 5%,
about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about
4.4%, about 4.3%, about 4.2%, about 4.1%, about 4%, about 3.9%,
about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about
3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%,
about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about
2.2%, about 2.1%, about 2%, about 1.9%, about 1.8%, about 1.7%,
about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about
1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%,
about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.
[0102] The cell culture incubator can be used in combination with
the culturing conditions described herein, for example, to isolate
specific cell populations, to induce changes in cells, to introduce
exogenous materials into cells, to determine biomarker expression,
and as a diagnostic tool for patients.
[0103] The methods of the invention can be used to increase, for
example, transfection and transduction efficiency in cells.
Transduction can be used, for example, to introduce a viral vector
in a cell. Viral nucleic acid delivery systems can use recombinant
viruses to deliver nucleic acids for gene therapy. Non-limiting
examples of viruses that can be used to deliver nucleic acids
include retrovirus, adenovirus, herpes simplex virus,
adeno-associated virus, vesicular stomatitis virus, reovirus,
vaccinia, pox virus, and measles virus.
[0104] Transfection methods that can be used with methods of the
invention include, for example, lipofection, electroporation,
calcium phosphate transfection, chemical transfection, polymer
transfection, gene gun, magnetofection, or sonoporation. The
transfection can be a stable or transient transfection. The
transfection can be used to transfect DNA plasmids, RNA, siRNA,
shRNA, or any nucleic acid. The plasmids can encode, for example,
green fluorescent protein (GFP), selectable markers, and other
proteins of interest. The selectable markers can provide resistance
to, for example, G418, hygromycin B, puromycin, and blasticidin.
The transfection method used with a method of the invention can
further introduce a vector system encoding the CRISPR-Cas9 system
into a cell.
[0105] A method of the invention can increase the transfection or
transduction efficiency by, for example, about 2-fold, about
3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold,
about 8-fold, about 9-fold, about 10-fold, about 12-fold, about
14-fold, about 16-fold, about 18-fold, about 20-fold, about
25-fold, about 30-fold, about 35-fold, about 40-fold, about
45-fold, about 50-fold, about 60-fold, about 70-fold, about
80-fold, about 90-fold, or about 100-fold.
Therapeutic Uses
[0106] Subjects can be, for example, elderly adults, adults,
adolescents, pre-adolescents, children, toddlers, infants. Subjects
can be non-human animals, for example, a subject can be a mouse,
rat, cow, horse, donkey, pig, sheep, dog, cat, or goat. A subject
can be a patient.
[0107] A method of the invention can be used to treat or diagnose,
for example, cancer in a subject. A method of the invention can be
used to identify a therapeutic, a biomarker, a genetic mutation, an
epigenetic marker, or a therapeutic target for cancer. A method of
the invention can also be used to develop a library or database of
genetic mutations found in cancer. A method of the invention can be
used for personalized medicine. A method of the invention can be
used to determine the effect of a therapeutic on a specific cell
type.
[0108] A method of the invention can be used, for example, to
enrich specific populations of cells or induce expression of
specific genes, for example, biomarkers or epigenetic markers. A
method of the invention can be used, for example, to affect the
potency of stem cells or somatic cells. For example, a method of
the invention can be used to test the ability of stem cells to go
from, for example, totipotent to, for example, pluripotent,
oligopotent, or unipotent.
[0109] The change in gene expression can affect, for example, cell
quantity, cell morphology, cell growth, cell motility, cell
invasion, or cell adhesion.
[0110] Genomic, proteomic, and metabolic analysis can be conducted
on the cultured cells to, for example, identify biomarkers that can
be used for development of cancer therapies, drug development,
cancer vaccines, cancer screening, diagnostics, personalized
antibody development, hematopoietic stem cell transplantation,
organ transplantation, or cardiovascular disease treatment.
[0111] Non-limiting examples of cancers that can be analyzed in a
method of the invention include: acute lymphoblastic leukemia,
acute myeloid leukemia, adrenocortical carcinoma, AIDS-related
cancers, AIDS-related lymphoma, anal cancer, appendix cancer,
astrocytomas, basal cell carcinoma, bile duct cancer, bladder
cancer, bone cancers, brain tumors, such as cerebellar astrocytoma,
cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,
supratentorial primitive neuroectodermal tumors, visual pathway and
hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt
lymphoma, carcinoma of unknown primary origin, central nervous
system lymphoma, cerebellar astrocytoma, cervical cancer, childhood
cancers, chronic lymphocytic leukemia, chronic myelogenous
leukemia, chronic myeloproliferative disorders, colon cancer,
cutaneous T-cell lymphoma, desmoplastic small round cell tumor,
endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma,
germ cell tumors, gallbladder cancer, gastric cancer,
gastrointestinal carcinoid tumor, gastrointestinal stromal tumor,
gliomas, hairy cell leukemia, head and neck cancer, heart cancer,
hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal
cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma,
kidney cancer, laryngeal cancer, lip and oral cavity cancer,
liposarcoma, liver cancer, lung cancers, such as non-small cell and
small cell lung cancer, lymphomas, leukemias, macroglobulinemia,
malignant fibrous histiocytoma of bone/osteosarcoma,
medulloblastoma, melanomas, mesothelioma, metastatic squamous neck
cancer with occult primary, mouth cancer, multiple endocrine
neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia,
nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma,
neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,
oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous
histiocytoma of bone, ovarian cancer, ovarian epithelial cancer,
ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet
cell, paranasal sinus and nasal cavity cancer, parathyroid cancer,
penile cancer, pharyngeal cancer, pheochromocytoma, pineal
astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary
blastoma, plasma cell neoplasia, primary central nervous system
lymphoma, prostate cancer, rectal cancer, renal cell carcinoma,
renal pelvis and ureter transitional cell cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers,
skin carcinoma merkel cell, small intestine cancer, soft tissue
sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,
throat cancer, thymoma, thymic carcinoma, thyroid cancer,
trophoblastic tumor (gestational), cancers of unknown primary site,
urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom macroglobulinemia, and Wilms tumor.
[0112] Cell surface marker molecules, such as EPCAM, CD133, EGFR,
HER2, or CD20 can be used to identify a population of cells. In
some embodiments, a combination of cell surface markers, or a cell
surface marker signature, can be used to identify a population of
cells. In some embodiments, a cell surface marker, and/or a cell
surface marker signature can be used in medical diagnosis.
[0113] Epigenetic markers that can be assessed using a method of
the invention include, for example, DNA methylation, cytosine
methylation, hydroxymethylation, histone methylation, lysine
acetylation, lysine methylation, arginine methylation, serine
phosphorylation, threonine phosphorylation, protein
phosphorylation, protein ubiquitination, protein sumoylation,
presence of 5-methylcytosine, histone H3 acetylation, or histone H4
acetylation.
[0114] Methods that can be used to determine the presence of
biological markers include, for example, qPCR, RT-PCR,
immunofluorescence, immunohistochemistry, western blotting,
high-throughput sequencing, ELISA, or mRNA sequencing.
[0115] Target cell subpopulations can be used for personalized
medicine. For example, CTCs and CSCs can be used for
chemosensitivity testing whereby chemotherapy regimens can be
tested on cultured CTCs. An assessment of the effects of
chemotherapy drugs on CTCs including, for example, cell viability
and cell division, can be done to determine the efficacy of a given
drug.
[0116] The methods of the present invention can be used to monitor
subject response to a given cancer therapy conducted by serial
monitoring of the subject's CTC population as treatment progresses.
Blood samples can be analyzed on a regular basis, before, during,
and after treatment to assess CTC viability.
[0117] The methods of the invention can be used to monitor subjects
who are currently in remission to investigate the potential of
cancer relapse. Serial testing of subject blood for CTCs can be
conducted on a regular basis to determine the potential or
likelihood for cancer relapse. In some cases, serial testing can
result in earlier detection of relapse. Serial testing can also be
used for long-term longitudinal studies.
[0118] A method of the invention can be used to collect data about
patients for patient stratification during clinical trials. For
example, the presence of a specific biomarker found in a patient's
CTCs can be used to place the patient in appropriate clinical trial
groups, or can be used as exclusion criteria for other clinical
trial groups.
[0119] The invention described herein can provide data that can be
used for a medical professional to treat a patient. Treatment of a
patient can include diagnosis, prognosis or theranosis. Diagnoses
can comprise determining the condition of a patient. Diagnosis can
be conducted at one time point or on an ongoing basis. For example,
a patient can be diagnosed with cancer. In another example, a
cancer patient who is in remission can be routinely screened to
determine if a cancer relapse has occurred. Prognosis can comprise
determining the outcome of a patient's disease, the chance of
recovery, or how the disease will progress. For example,
identifying CTCs of a certain type can provide information upon
which a prognosis can be based. Theranosis can comprise determining
a therapy treatment. For example, a patient's cancer therapy
treatment can include chemotherapy, radiation, drug treatment, no
treatment, or any combination thereof. A patient can be monitored,
for example by serial blood testing, to measure CTC populations
before, during and after a patient undergoes treatment. A positive
response to therapy can result in a decreased CTC viability and
lower division rates.
Computer Systems
[0120] A method of the invention can be used to, for example,
sequence, image, or characterize the collected target cell
subpopulations. Further methods can be found in PCT/US14/13048, the
entirety of which is incorporated herein by reference.
[0121] The invention provides a computer system that is configured
to implement the methods of the disclosure. The system can include
a computer server ("server") that is programmed to implement the
methods described herein. FIG. 4 depicts a system 400 adapted to
enable a user to detect, analyze, and process images of cells and
sequence cells. The system 400 includes a central computer server
401 that is programmed to implement exemplary methods described
herein. The server 401 includes a central processing unit (CPU,
also "processor") 405 which can be a single core processor, a multi
core processor, or plurality of processors for parallel processing.
The server 401 also includes memory 410 (e.g. random access memory,
read-only memory, flash memory); electronic storage unit 415 (e.g.
hard disk); communications interface 420 (e.g. network adaptor) for
communicating with one or more other systems; and peripheral
devices 425 which may include cache, other memory, data storage,
and/or electronic display adaptors. The memory 410, storage unit
415, interface 420, and peripheral devices 425 are in communication
with the processor 405 through a communications bus (solid lines),
such as a motherboard. The storage unit 415 can be a data storage
unit for storing data. The server 401 is operatively coupled to a
computer network ("network") 430 with the aid of the communications
interface 420. The network 430 can be the Internet, an intranet
and/or an extranet, an intranet and/or extranet that is in
communication with the Internet, a telecommunication or data
network. The network 430 in some cases, with the aid of the server
401, can implement a peer-to-peer network, which may enable devices
coupled to the server 401 to behave as a client or a server. The
microscope and micromanipulator can be peripheral devices 425 or
remote computer systems 440.
[0122] The storage unit 415 can store files, such as individual
images, time lapse images, data about individual cells, cell
colonies, or any aspect of data associated with the invention. The
data storage unit 415 may be coupled with data relating to
locations of cells in a virtual grid.
[0123] The server can communicate with one or more remote computer
systems through the network 430. The one or more remote computer
systems may be, for example, personal computers, laptops, tablets,
telephones, smart phones, or personal digital assistants.
[0124] In some situations the system 400 includes a single server
401. In other situations, the system includes multiple servers in
communication with one another through an intranet, extranet and/or
the Internet.
[0125] The server 401 can be adapted to store cell profile
information, such as, for example, cell size, morphology, shape,
migratory ability, proliferative capacity, kinetic properties,
and/or other information of potential relevance. Such information
can be stored on the storage unit 415 or the server 401 and such
data can be transmitted through a network.
[0126] Methods as described herein can be implemented by way of
machine (e.g., computer processor) computer readable medium (or
software) stored on an electronic storage location of the server
401, such as, for example, on the memory 410, or electronic storage
unit 415. During use, the code can be executed by the processor
405. In some cases, the code can be retrieved from the storage unit
415 and stored on the memory 410 for ready access by the processor
405. In some situations, the electronic storage unit 415 can be
precluded, and machine-executable instructions are stored on memory
410. Alternatively, the code can be executed on a second computer
system 440.
[0127] Aspects of the systems and methods provided herein, such as
the server 401, can be embodied in programming. Various aspects of
the technology may be thought of as "products" or "articles of
manufacture" typically in the form of machine (or processor)
executable code and/or associated data that is carried on or
embodied in a type of machine readable medium (e.g., computer
readable medium). Machine-executable code can be stored on an
electronic storage unit, such memory (e.g., read-only memory,
random-access memory, flash memory) or a hard disk. "Storage" type
media can include any or all of the tangible memory of the
computers, processors or the like, or associated modules thereof,
such as various semiconductor memories, tape drives, disk drives
and the like, which may provide non-transitory storage at any time
for the software programming. All or portions of the software may
at times be communicated through the Internet or various other
telecommunication networks. Such communications, for example, may
enable loading of the software from one computer or processor into
another, for example, from a management server or host computer
into the computer platform of an application server. Thus, another
type of media that may bear the software elements includes optical,
electrical, and electromagnetic waves, such as used across physical
interfaces between local devices, through wired and optical
landline networks and over various air-links. The physical elements
that carry such waves, such as wired or wireless likes, optical
links, or the like, also may be considered as media bearing the
software. As used herein, unless restricted to non-transitory,
tangible "storage" media, terms such as computer or machine
"readable medium" refer to any medium that participates in
providing instructions to a processor for execution.
[0128] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, tangible storage medium, a carrier wave medium, or
physical transmission medium. Non-volatile storage media can
include, for example, optical or magnetic disks, such as any of the
storage devices in any computer(s) or the like, such may be used to
implement the system. Tangible transmission media can include:
coaxial cables, copper wires, and fiber optics (including the wires
that comprise a bus within a computer system). Carrier-wave
transmission media may take the form of electric or electromagnetic
signals, or acoustic or light waves such as those generated during
radio frequency (RF) and infrared (IR) data communications. Common
forms of computer-readable media therefore include, for example: a
floppy disk, a flexible disk, hard disk, magnetic tape, any other
magnetic medium, a CD-ROM, DVD, DVD-ROM, any other optical medium,
punch cards, paper tame, any other physical storage medium with
patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM,
any other memory chip or cartridge, a carrier wave transporting
data or instructions, cables, or links transporting such carrier
wave, or any other medium from which a computer may read
programming code and/or data. Many of these forms of computer
readable media may be involved in carrying one or more sequences of
one or more instructions to a processor for execution.
Gas Flow Regulation System
[0129] Embodiments of the cell culture incubator include sensors
for atmospheric parameters such as oxygen, carbon dioxide, total
gas pressure, temperature, and dew point, each of which deliver
sensory data to the control unit. Within the control unit, these
data are received and acted upon by various atmospheric control
modules such as an oxygen module, a pressure module, a carbon
dioxide module, a temperature module, and a dew point module. By
way of various control pathways, these modules each engage features
and mechanics of a cell culture incubator gas flow system that
operate to establish the atmospheric parameters within the
incubators, such as individual gas sources, flow lines, flow
valves, pumps, and vents.
[0130] FIGS. 24A-24B depict embodiments of a gas flow system 2400
for a cell culture incubator that control the flow of various gases
into enclosed environment chamber 2401 in order to regulate aspects
of the gaseous or atmospheric environment within the chamber. These
parameters particularly include the total gas pressure and the
oxygen concentration of the total gas composition, but further may
include carbon dioxide, nitrogen, and water vapor. FIG. 24A is a
schematic diagram of an embodiment of gas flow control system 2400
to be used with a cell culture incubator and a method of the
invention. FIG. 24B is schematic diagram of an embodiment of a
nitrogen flow system 2450 (which may be considered a subsystem of
gas flow control system 2400) to be used with a cell culture
incubator and a method of the invention. Embodiments of the
invention, per systems as depicted in FIGS. 24A-24B, are directed
toward creating cell culture conditions that include both low
oxygen and high pressure, both parameters being regulated
independently of each other.
[0131] FIGS. 24A-24B show major components of system 2400 and 2450,
respectively, which include an incubator chamber 2401, a chamber
door 2416 (FIG. 24B) an incubator chamber heater 2402, an air
injection pump 2403, a recirculation pump 2404, a display 2405, and
a control unit 2406. Label 2401 refers to a cell culture
incubator's enclosed environmental chamber, but, for simplicity,
may also refer more generally to an incubator as a whole. Both
pumps 2403 and 2404 are in operative communication with the
interior of enclosed incubator chamber 2401. As described earlier
in the disclosure, embodiments of the cell culture incubator and
its control systems are typically controlled by way of user input
via a user interface (FIG. 15), and by automatic action of pumps
and valves by way of sensory feedback from within the incubator
chamber 2401, as mediated by control unit 2406 and its component
control modules, as described further below.
[0132] Gas flow into and out of enclosed environmental chamber 2401
by way of gas control system 2400 includes controlled input of
nitrogen gas 2434, controlled input of carbon dioxide 2432, and
controlled input of air 2436 (by way of injection pump 2403). The
internal atmosphere within the incubator also has an influx and
efflux by way of recirculation pump 2404, which facilitates a
homogeneous mixing of gases within enclosed environmental chamber
2401. Influx of nitrogen, carbon dioxide, and air, by way of their
respective pumps, is controlled by way of control unit 2406, by way
of sensors and gas control modules, as described below.
[0133] A number of types of sensors may be included within
incubator chamber 2401 that are responsive to various atmospheric
conditions, and which transmit sensed data to control unit 2406 and
its various control modules. These sensors include an oxygen sensor
2410, a carbon dioxide sensor 2411, a pressure sensor 2412, a
temperature probe 2413, a dew point sensor 2414. In some
embodiments of system 2400, one or more pressure sensors may be
included that are disposed and configured to measure external
ambient atmospheric pressure. Various types of oxygen sensors are
commercially available and suitable for embodiments of the
invention. For example, AMI (Huntington Beach, Calif.)
manufacturers an oxygen probe that delivers oxygen level data in
terms of concentration. Instruments that deliver concentration data
typically make use of a reference gas or reference to ambient air.
Another example of a suitable oxygen probe is provided by SST
(Coatbridge, UK), which delivers oxygen level data in terms of
partial pressure. Control unit 2406 (via display 2405) provides
oxygen level data in terms of concentration, even if sensor data
reports oxygen partial pressure. Most fundamentally, the basic
oxygen parameter is its partial pressure, which can be expressed
either directly or by conversion, or by comparison to reference
data, as a relative percent of a total atmospheric composition.
[0134] In a typical embodiment, data from dew point sensor is
directed by control unit 2406 to display 2405, and in some
embodiments, control of humidity is passive (as for example when
humidity is maintained by way of evaporation of liquid water in the
enclosed chamber) without the intervention of a dedicated control
module.
[0135] A contact sensor 2475 (FIG. 24B) may also disposed at a site
interfacing between chamber door 2416 that is sensitive to contact
between the door and its frame. In one embodiment, for example,
contact sensor 2475 is a depressable button that operates valve
2474. The role of valve 2474 in controlling gas input, particularly
nitrogen input, into incubator chamber 2401 is described further
below.
[0136] Within control unit 2406 are several control modules; these
include an oxygen module 2420, a carbon dioxide module 2422, a
pressure module 2424, and a temperature module 2426. Each of these
modules receive sensory input from their corresponding sensors,
i.e., oxygen sensor 2410, carbon dioxide sensor 2411, pressure
sensor 2412, and temperature probe 2413, respectively. Signals from
each of these types of sensor are received by corresponding modules
and used to formulate instructions that are sent to the various gas
flow control mechanisms and pumps, as described herein, to achieve
the instructed atmospheric parameters. Sensor values, or an
algorithm-derived expression thereof, may also be shown in display
2405, as exemplified by FIGS. 16-18.
[0137] Control unit 2406 effects control of the atmospheric
environment within enclosed environmental chamber 2401 by several
control paths. Display control path 2441 informs the read out on
display 2405. Heater path 2443 is responsive to temperature module
2426, and controls the operation of heater 2402. Recirculation pump
2404 operates at a constant rate that can be set, but is typically
not subject to sensory feedback control.
[0138] Gas control path 2442 is shown in a simplified depiction as
a single line, but it represents control paths for the operation of
nitrogen 2434 inflow, carbon dioxide 2432 inflow, vent efflux 2438
control, and air 2436 injection by way of air injection pump 2403.
The control of nitrogen inflow, carbon dioxide inflow, gas efflux
through the vent, and air inflow are all controlled more
particularly by valves or flow regulators that are not shown for
the sake of simplicity. Nitrogen 2434 inflow control is responsive
to oxygen module 2420 and pressure module 2424. Injection of
nitrogen may be used both to increase pressure within enclosed
environmental chamber 2401 and to decrease the oxygen
concentration, as described further below.
[0139] In typical operation, cell culture incubator 2401 operates
at an oxygen level that ranges from an ambient oxygen level to a
lower oxygen level, as described earlier. Although some embodiments
of incubator 2401 may be configured to operate at oxygen levels
higher than ambient levels, typical embodiments of incubator do
not. A typical operational task, therefore, is to decrease oxygen
concentration to a level less than that of the ambient condition.
This oxygen-lowering task is accomplished primarily by injection of
nitrogen 2434, which is controlled by control unit 2406 with input
from the oxygen module 2420. If the oxygen level drifts to level
higher than a targeted or instructed level, nitrogen injected into
enclosed environmental chamber 2401 mixes with existing gas
composition and drives the oxygen level by dilution. Once the
instructed oxygen level is achieved, control unit 2406 shuts off
nitrogen injection.
[0140] Referring now particularly to FIG. 24B, and returning to a
description of a gas driven safety lock mechanism 2472. For
orientation, it can be seen that as a gas such as nitrogen 2434
enters the incubator, it splits into two paths: a chamber gas flow
path 2460 and a door lock control path 2470. Chamber gas flow path
2460 includes intervention of regulator 2462 and valve 2464.
Details of the control of chamber gas flow path are covered above
in the description of gas control system 2400 as a whole, and as
shown in FIG. 24A.
[0141] The operation of a cell culture incubator at a
higher-than-ambient total gas pressure is benefited from a
construction that is fortified against gas leakage, and which
allows a door to the incubator to opened safely, without undue
disturbance of the atmosphere within the incubator, undue
disruption of gas regulation controls, and without unnecessary loss
of gas that being injected in to enclosed environmental chamber
2401. Accordingly, some embodiments of the cell culture incubator
include a door 2416 to the enclosed environmental chamber and a
safety lock 2472 configured to prevent opening of the door when the
enclosed environmental chamber is in a pressurized condition.
[0142] Some of these the safety lock embodiments 2472 include a
piston configured to be able to assume a locked position and an
unlocked position, the locked position of the safety lock being
secured by a gas pressure behind the piston, and the unlocked
position being assumed by a release of such gas pressure. In some
of these embodiments, the gas pressure is provided by nitrogen, the
nitrogen being delivered to a piston chamber behind the piston, the
nitrogen being provided to the chamber by way of a piston gas flow
control path 2470 from the nitrogen source. Use of nitrogen for the
purpose of driving door control path 2470 is a practical choice;
but oxygen or carbon dioxide, or any other gas being put into the
system would also work. Nitrogen is practical for this use because
it is already used in relatively high volume and because release of
nitrogen into the atmosphere is benign.
[0143] Tracking the elements of door lock control path 2470, it can
be seen that nitrogen 2434 encounters locking piston 2472 which is
configured to penetrate into enclosed environmental chamber door
2416 in its on position, the on position being secured by nitrogen
pressure behind locking piston 2472. A contact sensor 2475 (as
noted above in the context of enumerating the various sensors in
the system) is positioned at a point of contact between chamber
door 2416 and its enclosing frame, such that it senses whether the
door is open or closed, and communicates the open/closed status to
controller 2406.
[0144] During operation of enclosed environmental chamber 2401,
door 2416 is closed and locked by locking piston 2472. An opening
sequence begins with user input to allow the door to open, in
response, controller 2406 instructs valve 2474 to open, thereby
stopping the flow of nitrogen into a chamber behind the piston,
thereby releasing the pressure that supports the projection of the
piston into door 2416, thereby allowing the door to be manually
opened. In a locking sequence, contact sensor 2475 senses that the
door has been closed, the controller opens valve 2474, thereby
resuming the flow of nitrogen into the chamber behind the piston,
thereby driving the front portion of locking piston 2472 into door
2416, and securely locking it shut.
[0145] Central to the operation of enclosed environment chamber
2401 and to the mission of controlling atmospheric or gaseous
aspects of the environment for the purpose of creating particular
effects on cultured cells is the formation of high fidelity and
tunable atmosphere typically characterized by low oxygen (lower
than the ambient level) and high pressure (higher than the ambient
level), these two parameters being independently adjustable. It is
noteworthy that the goal of instilling high pressure, in and of
itself, inherently works against instilling low oxygen. Low oxygen
is commonly and reasonably expressed as a percent, as, for example,
can be read on display 2405, and as seen in FIGS. 16-18. In some
embodiments, oxygen sensor 2410 is actually sensing a partial
pressure of oxygen gas, an absolute term, not a relative % term. To
derive an oxygen percent concentration, control unit 2406 considers
both the partial pressure signal from oxygen sensor 2410 and either
a total gas pressure signal or a reference gas, and by way of a
formula delivers the "oxygen level %" value seen on display
2405.
[0146] In addition to oxygen partial pressure being the most basic
oxygen level parameter, partial pressure is also the oxygen
parameter of importance to cells in culture. Total gas pressure, by
itself, is also a highly important atmospheric parameter for cells
in culture, but it is separate from the effects of oxygen, as
measured in partial pressure terms. This description of the oxygen
level in terms of a fraction of the total gas pressure is being
provided because it makes it clear that increasing the total gas
pressure also, inherently, increases the partial pressure of any
component gas species within the total gas composition.
Accordingly, increasing total gas pressure increases the oxygen
partial pressure, which is working against the goal of decreasing
oxygen partial pressure. Accordingly, in embodiments of the
invention, and to the extent that total gas pressure works to
counter the goal of creating a low oxygen environment, instructions
from control unit 2406 to create a low oxygen condition prevail
despite the coincident instructions to create a high pressure
condition.
EXAMPLES
Example 1: Identification of Markers Associated With Prostate
Cancer
[0147] FIG. 5 shows the results of an experiment measuring
expression levels of cancer-associated proteins using samples of
CTCs obtained from a sample of patients with prostate cancer. The
CTCs were grown and cultured according to a method of the invention
to obtain an enriched CTC population. Gene expression of various
markers associated with prostate cancer was assessed using qPCR.
CD45 expression was assessed via immunofluorescence for two
subjects, 43-B and 45-C. The white arrow indicates staining for
CD45 and the black arrows indicate staining for EPCAM (epithelial
cell adhesion molecule)/PSMA. The top panel only contains CD45
staining. The markers assessed included androgen receptor (AR),
androgen receptor splice variant 7 (AR-V7), EPCAM, prostatic acid
phosphatase (PAPS), prostate-specific antigen (KLK3/PSA),
prostate-specific membrane antigen (FOLH1/PSMA), v-ets avian
erythroblastosis virus E26 oncogene homolog (ERG), prostate cancer
antigen 3 (PCA3), Nk3 homeobox 1 (NKX3-1), and chromogranin A or
parathyroid secretory protein 1 (CHGA).
[0148] The results indicated that PAPS was expressed in all
prostate tumor samples. The expression of other prostate cancer
markers differed among the samples indicating that CTC colonies can
be genetically diverse between subjects.
Example 2: Identification of Markers Associated With Prostate
Cancer
[0149] To identify immunotherapeutic targets and stem cell markers
that can be expressed by prostate CTC colonies, 10 to 20 mL of
peripheral blood was collected from over 30 subjects with
metastatic CRPC (mCRPC). Eight of the subject samples yielded CTC
colonies after culturing using a method of the invention as
described in FIGS. 2-3. Four of the samples were used for qPCR
analysis of several markers and were compared to the LNCaP
(prostate adenocarcinoma) and PC-3 (prostate adenocarcinoma) cell
lines as shows in FIG. 6, which contains the same staining pattern
described for FIG. 1.
[0150] A representative immunofluorescence image is shown with
DAPI, WBC, cytokeratin, and PSMA/PSA staining as in FIG. 1. The
results indicated that several of the patient samples and cell
lines expressed the immunotherapeutic targets programmed death
ligand 1 (CD274/PD-L1) and cytotoxic T-lymphocyte-associated
protein 4 (CTLA4) and the stem cell markers octamer-binding
transcription factor 4 (PUSF1/Oct4), SRY (sex determining region
Y)-box 2 (SOX2), keratin 18, type 1 (KRT18), and keratin 14, type 1
(KRT14), and that there was upregulation in Oct4, SOX2, and
PD-L1.
Example 3: RNA Sequencing of Prostate Cancer CTC Colonies
[0151] FIGS. 7-9 display results of a mRNA sequencing analysis to
determine differential expression of specific signaling pathways
among CTC colonies obtained from a sample of subjects. The CTC
colonies were obtained using a method as described in FIGS. 2-3.
The RNA sequence of the cells was mapped to specific genes, and the
gene counts were normalized across a collection of samples. Using a
non-parametric enrichment algorithm, statistical tests were
performed to detect pathways associated with relatively high
expression in each sample. False discovery rates were calculated
across large collections of pathways. The enrichment test results
were expressed as a false discovery rate on the x-axis for each
prostate sample RNA profile as seen in FIGS. 7-9. The enrichment
for gene expression in different pathways was different across the
samples. Pathways that show enrichment in prostate CTC colonies
included Nerve Growth Factor Receptor Signaling (FIG. 7), Aurora A
Signaling (FIG. 8), and Kit Receptor Signaling (FIG. 9)
Example 4: EPCAM Expression of Pancreatic CTC Colonies
[0152] To determine EPCAM expression in pancreatic CTC colonies, 6
patients with pancreatic ductal adenocarcinoma (PDAC) were
profiled. Apheresed blood samples were collected and cultured to
yield CTC colonies. The cells were stained for cytokeratin 19
(CK19; top left panel, and circular staining in right panel) and
EPCAM (punctate staining around cell in bottom left panel, and
peripheral staining indicated by white arrows in right panel) as
shown in FIG. 10. Cell binding to the collagen-based substrate used
for culturing of the CTCs led to increased expression of EPCAM.
Example 5: Mutations Exhibited by Pancreatic CTC Colonies
[0153] Six pancreatic CTC colony samples were analyzed for
mutations found in pancreatic cancer as determined from the COSMIC
(Catalogue of Somatic Mutations in Cancer) database. In the COSMIC
database for PDAC tumors, the mutation rate in KRAS is 69%, p53 is
51%, cyclin-dependent kinase inhibitor 2A (CDKN2A) is 23%, SMAD4 is
21%, AT-rich interactive domain-containing protein 1A (ARID1A) is
6%, and beta-catenin (CTNNB1) is 2%. For CTC colonies obtained
after culturing, 2/6 of the colonies displayed mutations in KRAS,
and 1/6 colonies displayed mutations in p53, CDK2NA, and
CTNNB1.
[0154] Example 6: Gene Expression in Pancreatic and mCRPC CTC
Colonies
[0155] To determine whether there was differential expression
between CTCs obtained from different tumors, PDAC CTCs were
compared to mCRPC CTCs. Pancreatic CTCs exhibited increased gene
expression in the NANOG, Wnt, insulin-like growth factor 1 (IGFR1),
FOXP1, and AR signaling pathways. The RNA sequence of the cells was
mapped to specific genes, and the gene counts were normalized
across a collection of samples. Using a non-parametric enrichment
algorithm, statistical tests were performed to detect pathways
associated with relatively high expression in each sample. False
discovery rates were calculated across large collections of
pathways. The enrichment test results were expressed as a false
discovery rate on the x-axis for each prostate sample RNA profile
as seen in FIGS. 11-12. The enrichment for gene expression in
different pathways was different across the samples. Pathways that
show enrichment in pancreatic CTC colonies over prostate cancer
CTCs included the NANOG signaling pathway (FIG. 11) and the Wnt
signaling pathway (FIG. 12). Dendritic cells, lung and T-cells were
used as controls, and the rest of the samples were CTCs obtained
from the subjects.
Example 7: SNP/INDEL Variant Analysis
[0156] To determine if CTC fractions displayed more genetic
variation than whole blood cell controls, single nucleotide
polymorphisms (SNPs) and insertions/deletions (INDELs) were
analyzed for six patients with stage 4 PDAC. FIG. 13 depicts the
results of SNP and INDEL analysis and indicates that both in terms
of total events (left panel) and total number of genes with events
(right panel), the CTC samples were able to uncover more genetic
variants compared to whole blood cell controls.
[0157] Using a targeted sequencing method, patient samples were
assessed for 238 genes associated with PDAC. FIG. 14 illustrates
the results of the sequencing analysis and shows that CTCs (left
side) revealed about 20-30 times more SNPs and INDELs compared to
whole blood cell controls for specific genes. The numbers at the
top of the chart denote the patient from which the sample was
obtained.
Example 8: User Interfaces for Displays in a System of the
Invention
[0158] FIG. 15 depicts a graphical interface that is seen by the
user upon initialization of a system of the invention. In the
top-left corner, the user enters his/her user identification code.
In the top-right corner, the user selects the desired option. The
quadrant at the bottom-left corner indicates the progress of the
desired operation. The bottom-right corner allows the user to enter
his/her password for the system. FIG. 16 depicts an additional user
interface that can be displayed by a system of the invention upon
logging into the system. The user interface of FIG. 16 can be
configured to be displayed, for example, for a minimum duration of
time, until the hardware connectivity is verified, and until the
user hits the start button.
[0159] FIG. 17 is a screen displaying the status of the oxygen
level (%), chamber pressure (PSI), temperature (.degree. C.), and
carbon dioxide level (%) in the culture chamber. The screen further
displays the relative humidity and experiment time remaining. The
user can further enter alarm settings and time settings using the
icons at the bottom of the screen. In this scenario, the oxygen
level was 20.0%, the chamber pressure was 3.7 PSIG, the temperature
was 34.2.degree. C., and the carbon dioxide level was 6.3%. The
relative humidity was 90%, and the experiment time remaining was 2
hours 38 minutes and 20 seconds.
[0160] FIG. 18 shows that upon selection of a specific parameter,
the user can touch the arrows and decrease or increase the
parameter to a desired value. The user can tap the arrow to change
the value in small increments, or hold down the arrow to change the
value at larger increments. Once the user has reached the desired
value, the user touches the value, and the desired value becomes
confirmed.
Example 9: Coating Process for Cell Adhesion
[0161] To prepare a surface for covalent binding of cellular
proteins, glass slides were prepared by incubating the slides with
0.1 M hydrochloric acid (HCl) for two hours to overnight at room
temperature. Then, the glass slides were incubated with 0.1 M NaOH
for two hours to overnight at room temperature. The slides were
then incubated with 0.5-5% (3-aminopropyl)-trimethoxysilane for two
hours to overnight at room temperature. Then, the slides were
incubated with 0.5-5% glutaraldehyde (diluted in PBS) for 2 hours
to overnight at room temperature. The glass slides were then rinsed
with water overnight, sterilized under UV light for one hour, and
then stored dry at room temperature.
[0162] The slides were then further treated to facilitate cell
binding using a mixture containing extracellular matrix (ECM)
proteins. The ECM mix contained from about 0.1 to about 3 mg/mL
collagen, from about 0.1 to about 10 .mu.g/mL fibronectin, and from
about 0.1 to about 10 .mu.g/mL of a basement membrane cocktail. The
ECM mix was diluted with either a glycine buffer at pH 10 or a DMEM
buffer at pH 7 depending on the cellular application.
Example 10: Transfection Efficiency Using a Method of the
Invention
[0163] DU145 (human prostate cancer) cells were transfected with a
GFP plasmid using electroporation. 5.times.106 cells/mL were
electroporated using a protocol of 1260 V for 20 ms twice with 50
ng DNA plasmid/.mu.t of the cell resuspension. After transfection,
the cells were split into separate 35 mm cell culture plates. One
plate was placed in a standard CO2 incubator, and the other plate
was place in an incubator of the invention. The second plate's
incubator was set to 1% O2 and 2 PSIG. After 48 hours, the cells
were fixed and imaged for GFP expression using a fluorescent
microscope. The data shown in FIG. 23 indicate that cells incubated
at low oxygen and high pressure showed higher transfection
efficiency (lower panel) than those cells incubated at standard
conditions (upper panel) as depicted by a greater number of bright
cells.
EMBODIMENTS
[0164] The following non-limiting embodiments provide illustrative
examples of the invention, but do not limit the scope of the
invention.
Embodiment 1
[0165] A cell culture incubator, wherein the cell culture incubator
comprises: a) an enclosed environmental chamber; and b) a control
unit, wherein the control unit is operably linked to the enclosed
environmental chamber, wherein the control unit comprises a
computer program product comprising a computer-readable medium
having computer-executable code encoded therein, the
computer-executable code adapted to encode: (i) an oxygen level
module, wherein the oxygen level module is encoded to regulate an
oxygen level of the enclosed environmental chamber, wherein the
oxygen level module is encoded to control the removal of oxygen in
the enclosed environmental chamber to generate a hypoxic oxygen
level within the enclosed environmental chamber; (ii) a pressure
module, wherein the pressure module is encoded to regulate a
pressure of the enclosed environmental chamber, wherein the
pressure module controls the addition of gas to generate a positive
pressure condition in the enclosed environmental chamber; (iii) a
temperature module, wherein the temperature module is encoded to
regulate a temperature of the enclosed environmental chamber; and
(iv) a humidity module, wherein the humidity module is encoded to
regulate a humidity of the enclosed environmental chamber, wherein
each of the oxygen level, pressure, temperature, and humidity
mimics an in vivo microenvironment for a cell, wherein the cell
culture incubator reaches each of an instructed oxygen level,
pressure, temperature, and humidity within about 20 minutes of
receiving an input of each of the instructed oxygen level,
pressure, temperature, and humidity.
Embodiment 2
[0166] The cell culture incubator of embodiment 1, wherein the in
vivo microenvironment is a tumor microenvironment.
[0167] Embodiment 3
[0168] The cell culture incubator of any one of embodiments 1-2,
wherein the cell is a stem cell.
Embodiment 4
[0169] The cell culture incubator of any one of embodiments 1-2,
wherein the cell is a cancer cell.
Embodiment 5
[0170] The cell culture incubator of any one of embodiments 1-2,
wherein the cell is a circulating tumor cell.
Embodiment 6
[0171] The cell culture incubator of any one of embodiments 1-2,
wherein the cell is an immune cell.
Embodiment 7
[0172] The cell culture incubator of any one of embodiments 1-6,
wherein the cell is obtained from a biological sample.
Embodiment 8
[0173] The cell culture incubator of embodiment 7, wherein the
biological sample is blood.
Embodiment 9
[0174] The cell culture incubator of embodiment 7, wherein the
biological sample is a tumor.
Embodiment 10
[0175] The cell culture incubator of embodiment 7, wherein the
biological sample is saliva.
Embodiment 11
[0176] The cell culture incubator of embodiment 7, wherein the
biological sample is a tissue.
Embodiment 12
[0177] The cell culture incubator of any one of embodiments 1-11,
wherein the control unit is user-controlled.
Embodiment 13
[0178] The cell culture incubator of any one of embodiments 1-11,
wherein the control unit is automated.
Embodiment 14
[0179] The cell culture incubator of any one of embodiments 1-13,
wherein the oxygen module is encoded to maintain a hypoxic oxygen
level.
Embodiment 15
[0180] The cell culture incubator of any one of embodiments 1-14,
wherein the pressure module is encoded to maintain a positive
pressure condition.
Embodiment 16
[0181] The cell culture incubator of any one of embodiments 1-15,
wherein the oxygen level module is encoded to maintain the oxygen
level in the enclosed environmental chamber at about 0.1% to about
21%.
Embodiment 17
[0182] The cell culture incubator of any one of embodiments 1-16,
wherein the oxygen level module is encoded to maintain the oxygen
level in the enclosed environmental chamber at about 2%.
Embodiment 18
[0183] The cell culture incubator of any one of embodiments 1-17,
wherein the oxygen level module is encoded to maintain the oxygen
level in the enclosed environmental chamber at about 0.1%.
Embodiment 19
[0184] The cell culture incubator of any one of embodiments 1-18,
wherein the pressure module is encoded to maintain the pressure in
the enclosed environmental chamber at from about 1 PSIG to about 5
PSIG.
Embodiment 20
[0185] The cell culture incubator of any one of embodiments 1-19,
wherein the humidity module is encoded to maintain the humidity in
the enclosed environmental chamber at about 85%.
Embodiment 21
[0186] The cell culture incubator of any one of embodiments 1-20,
wherein the control unit further comprises a computer program
product comprising a computer-readable medium having
computer-executable code encoded therein, the computer-executable
code adapted to encode a carbon dioxide module, wherein the carbon
dioxide module is encoded to regulate a carbon dioxide level of the
enclosed environmental chamber.
Embodiment 22
[0187] The cell culture incubator of any one of embodiments 1-21,
wherein the cell culture incubator further comprises a gas inlet
controlled by the oxygen level module.
Embodiment 23
[0188] The cell culture incubator of any one of embodiments 1-22,
wherein the cell culture incubator further comprises a gas inlet
controlled by the pressure module.
Embodiment 24
[0189] The cell culture incubator of any one of embodiments 1-23,
wherein the cell culture incubator further comprises a water
humidity tray controlled by the humidity module.
Embodiment 25
[0190] The cell culture incubator of any one of embodiments 1-24,
wherein the cell culture incubator further comprises a heating
element controlled by the temperature module.
Embodiment 26
[0191] The cell culture incubator of any one of embodiments 1-25,
wherein the cell culture incubator is configured to accept a cell
culture plate.
[0192] Any one or more features of any embodiment of the inventions
disclosed herein may be combined with any one or more other
features of any other embodiment of the inventions, without
departing from the scope of the invention. It should also be
understood that while some theoretical considerations may have been
provided to further an understanding of embodiments of the
invention, the claims to the invention are not bound by such
theory. It should also be understood that the invention is not
limited to the embodiments that are described or depicted herein
for purposes of exemplification, but is to be defined only by a
fair reading of claims appended to the patent application,
including the full range of equivalency to which each element
thereof is entitled.
* * * * *