U.S. patent application number 15/785011 was filed with the patent office on 2018-03-22 for cell culture medium.
The applicant listed for this patent is BioMarker Ltd.. Invention is credited to Henriette SKRIBEK, Laszlo SZEKELY.
Application Number | 20180080006 15/785011 |
Document ID | / |
Family ID | 41459954 |
Filed Date | 2018-03-22 |
United States Patent
Application |
20180080006 |
Kind Code |
A1 |
SKRIBEK; Henriette ; et
al. |
March 22, 2018 |
CELL CULTURE MEDIUM
Abstract
The present invention relates to a cell culture medium for
culturing human cells comprising an anti-coagulated total blood
material wherein the hemoglobin level is from about 8 to about 16
g/dl. More particularly, the invention provides a cell culture
medium in which cells present in the blood are disrupted and the
insoluble remnants of the lysated cells are removed. Further, the
invention provides a method for the preparation of a cell culture
medium for culturing human cells, according to the invention.
Inventors: |
SKRIBEK; Henriette;
(Godollo, HU) ; SZEKELY; Laszlo; (Varmdo,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioMarker Ltd. |
Godollo |
|
HU |
|
|
Family ID: |
41459954 |
Appl. No.: |
15/785011 |
Filed: |
October 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13497707 |
Nov 12, 2012 |
9790464 |
|
|
PCT/IB2010/054303 |
Sep 24, 2010 |
|
|
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15785011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2502/11 20130101;
C12N 2500/84 20130101; C12N 5/0018 20130101; C12N 5/0602
20130101 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/00 20060101 C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 2009 |
HU |
P0900603 |
Claims
1. A cell culture medium for culturing human cells comprising an
anti-coagulated total blood material wherein the hemoglobin level
is from about 8 to about 16 g/dl.
2. The cell culture medium according to claim 1, wherein the cells
present in the blood are disrupted or lysed and the insoluble
remnants of the disrupted or lysed cells are removed.
3. The cell culture medium according to claim 1, wherein the blood
is of mammalian origin.
4. The cell culture medium according to claim 3, wherein the blood
is of human origin.
5. The cell culture medium according to claim 1, wherein the ion
content of the medium is restored to match that of the
extracellular fraction of total blood starting material.
6. The cell culture medium according to claim 1, wherein the low
molecular weight (<3000 Dalton) nutrient content and/or
composition is essentially the same as that of a medium selected
form the group consisting of Roswell Park Memorial Institute (RPMI)
medium, Dulbecco's Modified Eagle's Medium (DMEM), and Isocove's
Modified Dulbecco's Medium (IMDM).
7. The cell culture medium according to claim 1, wherein the
reducing capacity of the medium is restored by addition of reduced
glutathione.
8. (canceled)
9. A method for the preparation of the cell culture medium for
culturing human cells of claim 1, said method comprising (a)
providing anti-coagulated mammalian blood as starting material; (b)
mechanically disrupting or lysing the cells present in the blood;
(c) removing the insoluble remnants of the disurupted or lysed
cells.
10. The method according to claim 9, wherein the mechanical
disruption of the cells is achieved by freezing the total blood or
by ultrasound, electric blade homogenizer or French press.
11. The method according to claim 9, wherein removal of the
insoluble remnants of the lysated cells is achieved by
ultracentrifugation or mechanical filtration.
12. The method according to claim 9, wherein the low molecular
weight (<3000 Dalton) content and/or composition of the medium
is adjusted by dialysis against conventional cell culture medium
selected form the group consisting of RPMI, DMEM, and IMDM.
13. The method according to claim 9, wherein the reducing capacity
of the medium is restored by addition of reduced glutathione.
14. Cell culture medium for culturing human cells, obtainable by
the method according to claim 9.
15. (canceled)
16. The cell culture medium according to claim 1, wherein said
culture medium is prepared by (a) obtaining mammalian blood having
an extracellular fraction, as starting material; (b) adding an
anticoagulant to the mammalian blood thereby providing
anti-coagulated mammalian blood; (c) mechanically disrupting or
lysing the cells present in the anti-coagulated mammalian blood;
(d) removing insoluble remnants of the disrupted or lysed cells to
produce a particle-free extract; (e) modifying the ion content of
the particle-free extract to match the extracellular fraction of
the starting material or to be similar to that of a medium selected
form the group consisting of Dulbecco's Modified Eagle's Medium
(DMEM), Roswell Park Memorial Institute (RPMI) medium and Isocove's
Modified Dulbecco's Medium (IMDM).
17. The cell culture medium according to claim 16, wherein the
growth and survival factor flora of the starting material is
preserved in the culture medium.
18. The cell culture medium according to claim 16, wherein the
anticoagulant is heparin.
19. The cell culture medium according to claim 16, wherein the
disrupting or lysing of the cells achieves an even disruption of
cell membranes of the cells.
20. The cell culture medium according to claim 16, wherein the
culturing medium contains all macromolecular components of the
blood.
21. The cell culture medium according to claim 16, wherein the
mechanical disruption or lysing of the cells is achieved by
freezing the total blood.
Description
[0001] This is a divisional of application Ser. No. 13/497,707, now
U.S. Pat. No. 9,790,464, which is the national stage of
international application PCT/IB2010/054303, filed Sep. 24, 2010,
the entire disclosure of which is hereby incorporated by reference
herein.
[0002] The present invention relates to a cell culture medium for
culturing human cells comprising an anti-coagulated total blood
material wherein the hemoglobin level is from about 8 to about 16
g/dl. More particularly, the invention provides a cell culture
medium in which cells present in the blood are disrupted and the
insoluble remnants of the lysated cells are removed. Further, the
invention provides a method for the preparation of a cell culture
medium for culturing human cells, according to the invention.
BACKGROUND OF THE INVENTION
[0003] Culturing primary human cell of both normal and malignant
origin is a highly desirable but difficult task. The major reason
is that most defined culture media were selected to maintain a
well-defined specific cell type. These media are although rich in
all necessary low molecular weight compounds they often lack
critical growth and survival factors. Formulating cell culture
media often required that it should be possible to produce in very
large quantities for relatively low price. However recent
introduction of new microfluidic and automated microscopy
techniques make these requirements obsolete.
[0004] Primary cultures of normal cells can serve as a source for
stem cells for e.g. reconstitution therapy or as ex vivo target for
gene delivery in gene therapeutic interventions. Primary cultures
of tumor cells may be used for large scale drug sensitivity assays
in order to identify suitable anti-cancer drugs for a given
patient, test the efficacy of new drug candidates to previously
untested tumor types and even in screening of compound libraries to
identify new anti-cancer lead candidates.
[0005] An important feature of human tumors is that they are
continuously evolving and the ideal therapy would require
individualized measures perfectly matching the biological features
of the tumor. The most straightforward way to achieve this goal is
to create a sophisticated albeit highly robust and reliable methods
that can measure the response-rate of the tumor to a large variety
of therapeutic interventions. These measurements should provide the
theoretical underpinning and practical evidence for assay-guided
therapy.
[0006] Modern day treatment protocols are the results of carefully
controlled clinical trials involving thousands of patients over
many years. During the last decades, we have witnessed an
impressive improvement in patient survival in several forms of
leukemias and lymphomas. The development was the most impressive in
the case of pediatric leukemias. For these diseases, the careful
grouping of patients according different biological markers and
prognostic indicators proved to be crucial for selection of the
most effective treatment protocols. These protocols heavily rely on
the use of relatively old drugs developed in the late 70s and early
80s. Key factor to achieve the greatly improved survival was the
rigorous adherence to the standardized treatment protocol. This
strategy, albeit very successful in terms of saving lives of
pediatric patients, resulted in very aggressive treatment with
significant toxicity. Moreover, this strategy left little room for
introduction of new drugs. This is particularly unfortunate
considering that there are more than sixty licensed drugs with
potential anti-cancer effects and there are over hundred novel
drugs in the development pipeline of the pharmaceutical
industry.
[0007] On the other hand, in the case of adult leukemias, the major
breakthrough originated from the introduction of novel drugs to
treat near terminal patients (e.g. bcr-abl inhibitor Gleevec
against CML; proteasome inhibitor Bortezomib against multiple
myelomas or monoclonal antibodies such as anti CD20--Rituximab
against a variety of B-cell tumors). Despite all effort, a sizable
fraction of both pediatric and adult patients does not respond to
the therapy or experience repeated relapses. In these cases,
selection of individually designed treatment protocol and
application of novel drugs would be highly justified.
[0008] The idea to use in vitro drug sensitivity assays to
determine the sensitivity profile of leukemic cells was already
introduced forty years ago. A variety of techniques were developed
that aimed to determine the viability of tumor cells after in vitro
culturing with relevant drugs [1]. All assays involve the same four
basic steps: i) isolation of cells; ii) incubation of cells with
drugs in tissue culture medium; iii) assessment of cell survival;
iv) interpretation of the results. Some of these techniques are
widely used today such as the ATP conversion, MTT reduction
(tetrazolium salt conversion to insoluble formazan) or FMCA
(fluorometric microculture cytotoxicity) assays [2, 3] and has been
proven to provide clinically useful information about the drug
sensitivity of the tumor cells.
[0009] The current assays though suffer a number of limitations.
One of the most serious limitations is the difficulty to keep
primary human tumor cells alive in vitro [4]. Most eukaryotic cells
require two types of signals simultaneously for efficient ex vivo
expansion: growth-promoting (proliferation) and survival
(anti-apoptotic) signals. These signals are conferred as soluble
ligands, alternatively ligands that are part of the extracellular
matrix or are associated with the surface of neighboring cells. The
signaling molecules target receptors on the cells that are specific
for any given cell type and differentiation stage. Classical cell
culture media normally has to be supplemented by serum or tissue
extracts in order to support efficient in vitro cell growth. The
regularly used 10% fetal bovine serum provide sufficient amount of
PDGF and insulin like growth factors to support the expansion of
primary fibroblasts and mesenchymal stem cells. Most primary cells,
however, require the addition of extra growth factors. The
generation of in vitro adapted cell lines is achieved by selection
of rare genetic variants that are able to expand solely with the
help of PDGF and IGF like molecules. Both normal and tumor cells of
the lymphoid/myeloid lineage require extra growth/survival factors
for in vitro expansion. Importantly shortage of survival factors
sensitizes the cells for cytotoxic drug effects creating an
undesirable overestimation of the drug efficacy in short term in
vitro survival assays. The present day assays often require a
sizable amount of tumor cells and they are often very
labor-intensive. Due to the limited number of measurements they can
carry out, most assays do not allow testing of large number of
drugs in different concentrations in repeated series. Most assays
have low information content and regularly restricted to single
measurement readout (e.g. estimation of the number of live cells
per sample).
[0010] Moreover, not all drugs are suited for the conventional in
vitro survival assays. Some of them, such as the highly fluorescent
anthracyclines, are incompatible with direct fluorescence readout
technology. Despite of these shortcomings short-term survival
assays already started to deliver clinically important information
[1, 5-7].
[0011] Until recently, in vitro assays were regarded as an
unreliable approach to predict in vivo drug response. These
sentiments stemmed from the unimpressive results of in vitro
clonogenic assays of ovarium carcinomas. However, the perspective
of in vitro assays is shifting in the prediction of drug response,
as is evidenced by in a recent review [8] of most of the work,
concerning ex vivo drug sensitivity assays, published through the
1980s--"reflected the prevailing view of cancer as a disease of
dysregulated cell proliferation. The description of apoptosis and
programmed cell death, fundamental to our modern understanding of
human tumor biology, did not occur until well after the heyday of
in vitro chemosensitivity testing. By incorporating the modern
tenets of carcinogenesis associated with perturbations in cell
survival we can now re-examine laboratory assays of drug response
in the context of drug-induced programmed cell death."
[0012] The main reason for failures was that most cell culture
media were developed by starting out from chemically defined simple
solutions and gradually adding different serum or tissue extracts
until the test cells showed satisfactory survival and
proliferation. It is clear now that new approaches are necessary to
revive in vitro chemosensitivity testing, to allow finding the
optimal, and preferably personalized, treatment for cancer.
[0013] The rapid development of digital imaging, the recent
breakthroughs in microscopy and laser optics and new methods in lab
automation and microfluidics brought this idealized method within
reach. We have shown that it is now possible to build and program
automated intelligent confocal microscopes that can test the effect
of all presently available anti-cancer drugs on primary tumor cells
isolated from volumes as small as 1 ml blood, bone marrow, ascites
fluid and pleural effusion. The ability to manufacture 384 well
drug plates using preprogrammed fluid dispensing robots allows
testing of a large number of drugs in different concentrations,
alone or in different combinations. Sophisticated fluorescence
labeling techniques reveal multiple vital parameters of the drug
treated tumor cells such as viability, cell cycle distribution,
metabolic activity and motility.
[0014] The only missing element for comprehensive in vitro drug
testing is a reliable, efficient culturing medium that preserves
the original in vivo conditions as much as possible to help
identify the best possible treatment regimen.
[0015] To satisfy this need, we have completely re-designed the
culture conditions for primary tumor cells. In our approach, we
preserve the original in vivo conditions as much as possible. The
theoretical basis of our new cell culture medium relies on the
following previous observations: primary human lymphocytes, highly
apoptosis prone when explanted into cell culture media in the
absence of extra growth factors, can survive in total blood for
several days [9-12] even if it undergoes partial hemolysis [13].
However, none of these prior art discloses or even suggests the
feasibility whole blood as the basis of culture medium for
culturing human cells.
[0016] Accordingly, the present invention provides a cell culture
medium for culturing human cells comprising an anti-coagulated
total blood material wherein the hemoglobin level is from about 8
to about 16 g/dl.
[0017] In another embodiment, the invention provides a cell culture
medium, wherein the cells present in the blood are disrupted and
the insoluble remnants of the lysated cells are removed.
[0018] In a further embodiment, the invention provides a cell
culture medium, wherein the blood is of mammalian origin. In a
specific embodiment, the invention provides a cell culture medium,
wherein the blood is of human origin.
[0019] In another embodiment, the invention provides a cell culture
medium, wherein the ion content of the medium is restored to match
that of the extracellular fraction of total blood starting
material.
[0020] In a further embodiment, the invention provides a cell
culture medium, wherein the low molecular weight (<3000 Dalton)
nutrient content and/or composition is essentially the same as that
of a conventional cell culture medium selected preferably form the
group consisting of RPMI, DMEM, and IMDM.
[0021] In another embodiment, the invention provides a cell culture
medium, wherein the reducing capacity of the medium is restored by
addition of reduced glutathione.
[0022] In a further aspect, the invention provides the use of a
cell culture medium according to the invention for culturing human
cells.
[0023] In a further aspect, the invention provides a method for the
preparation of a cell culture medium for culturing human cells,
comprising
[0024] (a) providing anti-coagulated mammalian blood as starting
material;
[0025] (b) mechanically disrupting the cells present in the
blood;
[0026] (c) removing the insoluble remnants of the lysated
cells.
[0027] In another embodiment, the invention provides a method,
wherein the mechanical disruption of the cells is achieved by
freezing the total blood or by ultrasound, electric blade
homogenizer or French press.
[0028] In another embodiment, the invention provides a method,
wherein removal of the insoluble remnants of the lysated cells is
achieved by ultracentrifugation or mechanical filtration.
[0029] In another embodiment, the invention provides a method,
wherein the low molecular weight (<3000 Dalton) content and/or
composition of the medium is adjusted by dialysis against
conventional cell culture medium selected preferably form the group
consisting of RPMI, DMEM, and IMDM.
[0030] In another embodiment, the invention provides a method,
wherein the reducing capacity of the medium is restored by addition
of reduced glutathione.
[0031] In a further aspect, the invention provides a cell culture
medium for culturing human cells, obtainable by the method
according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The methods of establishment of primary cell cultures from
human tissues suffer number of limitations. In vitro explanted
primary cells require an environment that has similar
physico-chemical properties as well as an array of biological
signals that are similar to the in vivo milieu.
[0033] The present invention embodies a new cell culture medium
that maintains the correct ion, osmotic, and redox conditions of
the total blood along with fully preserved growth and survival
factor flora. The new cell culture medium is based on total blood
extract that contains all macromolecular components of blood in
addition to all small molecular nutrients of conventional tissue
culture medium but lacks the membrane fraction of the red blood
cells. We claim that the new medium is superior to conventional
media in supporting the survival of primary human cells of normal
and malignant origin. It is particularly suitable for drug
sensitivity testing of freshly explanted human tumor cells thus
creating the basis for a new technological platform for
determination of cytotoxic drug sensitivity profiles of individual
patients. Short-term drug sensitivity assay may pave the way for
individualized assay guided therapy of cancer. The optical
properties of the new medium permit fluorescence high content
analysis (HCA) evaluation of the treated cells. The medium also
quenches the autofluorescence of most of the commonly used drugs.
The medium is also well suited for high throughput screening of
large chemical of biological compound libraries or large scale
toxicological testing on primary human cells.
[0034] We have therefore developed, based on our empiric data, a
new cell culture medium that unites the cell survival promoting
components with the nutritional values of cell culture media in the
correct ion and redox environment. We have tested the medium on a
large series of cell lines of different origin, as well as on
primary tumors from ascites fluids or pleural effusions
accompanying ovarium, breast, colon, rectum, pancreas and bladder
carcinomas and on isolated tumor cells from AML, ALL, CLL, body
cavity lymphomas, Hodgkin and post-transplant lymphomas. When
comparing the new medium with conventional cell culture media, we
have regularly found improved survival and decreased cytotoxic drug
sensitivity of primary tumor cells. Our novel cell culture medium
permits extended survival of human tumor cells in vitro. Using the
new culture medium it is now possible to carry out assays that can
determine the drug sensitivity pattern of freshly isolated primary
tumor cells from patients suffering from leukemias, lymphomas or
advanced disseminated carcinomas.
[0035] The present invention comprises a procedure that converts
human (or animal) whole blood into a cell culture medium that
support the ex vivo survival of primary human cells and compatible
with various fluorescence imaging techniques for viability
determination and measurement of various physiological functions.
It is based on mechanical lysis of the undiluted anti-coagulated
blood, removal of red blood cell ghosts, restoration of the correct
ion content, equilibration with low molecular weight nutrients and
restoration of reducing conditions. The final product can be
immediately used in short-term assays or stored in frozen form over
a long period of time.
[0036] Although lysates of total blood is a frequent additive of
certain bacterial culture media such as blood agar, it is not used
in culturing of human or other mammalian cells. The current
invention concerns the development of eukaryotic cell culture
medium, primarily for ex vivo culturing of human tumor cells/stem
cells. The chemical composition of bacterial and eukaryotic media
is so much different that mammalian cells do not survive in that
bacterial medium. Since the whole blood is only a specific additive
in those media, the hemoglobin concentration is well below the
level which characterizes the present cell culture medium.
[0037] Accordingly, the present invention provides a cell culture
medium for culturing human cells comprising an anti-coagulated
total blood material wherein the hemoglobin level is from about 8
to about 16 g/dl. Given the fact that the present medium is
essentially composed of the whole blood starting material, the high
hemoglobin content is the result of the lysis of red blood cells
during the processing of the whole blood material. More
particularly, the hemoglobin concentration of the cell culture
medium according the invention is from about 10 to about 14 g/dl,
preferably from about 11 to about 12 g/dl, and even more preferably
about 11 g/dl.
[0038] Another advantageous characteristic of the cell culture
according to the invention is that although the medium per see is
not transparent, high throughput microscopic imaging can be
efficiently carried out. According to the state of the art, high
sensitivity fluorescence measurements of cell viability are not
compatible with several commonly used anticancer drugs (e.g.
epirubicin, daunorubicin, doxorubicin) with high endogenous
autofluorescence properties using conventional cell culture media.
We showed that using blood extract based culture medium the
disturbing autofluorescence is efficiently quenched. The present
cell culture medium is not transparent, having the characteristic
color of the blood, said high throughput microscopic imaging can be
carried out using inverted microscope systems where the cells are
collected at the bottom of the culture vessels.
[0039] A main feature of the cell culture medium according to the
invention is that the cellular components of the whole blood are
eliminated as such, but the materials contained said cellular
components are retained in the cell culture medium. This is
achieved by the disruption of the cells present in the blood and
the removal of the insoluble remnants of the lysated cells. Such
disruption and removal can be carried out by any suitable means
available to the person skilled in the art.
[0040] The present invention also provides a method for the
preparation of a cell culture medium for culturing human cells,
comprising
[0041] (a) providing anti-coagulated mammalian blood as starting
material;
[0042] (b) mechanically disrupting the cells present in the
blood;
[0043] (c) removing the insoluble remnants of the lysated
cells.
[0044] En exemplary protocol for the method of the invention is
given in Example 1, however, the person skilled in the art will be
readily capable to substitute different procedures to achieve
essentially the same results as given in the above steps. For
example, mechanical disruption of the cells can be achieved, as
non-limiting examples, by freezing the total blood or by
ultrasound, electric blade homogenizer or French press. The
optimization of the suitable parameters for each of these
techniques is also apparent for the person skilled in the art.
Similarly, after the cell disruption, removal of the cell ghosts
can be done by using one or more of several different state of the
art techniques. Non-limiting examples are ultracentrifugation or
mechanical filtration.
[0045] As we detailed above, one of the main objective to be
achieved by providing the cell culture medium according to the
invention is to ensure optimal culturing conditions for human cells
ex vivo. It is apparent that human blood will provide the closest
possible match for human cell culturing. Accordingly, as far as it
is feasible and possible, the blood base material for the present
cell culture medium is of human origin. Alternatively, when the
desired culturing protocol allows it or calls for, the human blood
based medium can be substituted for by a cell culture medium
according to the invention that is based on the whole blood of
another mammalian species, preferably of bovine or porcine
origin.
[0046] As it is apparent for the person skilled in the art, the
disruption of the cellular components of the whole blood will
release their content into the medium, altering the original
composition of the whole blood. In preferred embodiments,
therefore, it is also may be desired to modify the composition of
the resulting medium to more closely match the composition of the
starting material, i.e. the whole blood. Or alternatively, to make
its composition similar to the composition of cell culture media
conventionally used in the field. The person skilled in the art
will be readily able to determine what type of adjustment is the
most suitable for the intended use of the cell culture medium
according to the invention.
[0047] Accordingly, in a specific embodiment, the ion content of
the medium is restored to match that of the extracellular fraction
of total blood starting material. In another specific embodiment,
the reducing capacity of the medium is restored by addition of
reduced glutathione. Further, in another specific embodiment, the
low molecular weight (<3000 Dalton) nutrient content and/or
composition is essentially the same as that of a conventional cell
culture medium selected preferably form the group consisting of
RPMI, DMEM, and IMDM. The person skilled in the art will be able
the select from the available state of the art techniques to
achieve any of these modifications, or any other modification that
he deems appropriate. Non-limiting examples are given below in the
Examples.
DESCRIPTION OF THE FIGURES
[0048] FIG. 1: Schematic view of the production of blood extract
based cell culture medium
[0049] FIG. 2A: Design of basic 384-well plate for testing 30
drugs.
[0050] FIG. 2B: Primary human ovarian carcinoma cells after 7 days
culturing in blood extract based medium (Phase contrast--nuclear
staining with live cell compatible Hoechst 33342).
[0051] FIG. 2C: Sensitivity of DLBL cells to 30 different drugs as
analysed by individual imaging of 500.000 cells from 7680 confocal
sections.
[0052] FIG. 2D: Maximum concentrations of the drugs used.
[0053] FIG. 3: Drug sensitivity pattern of 38 different primary
human carcinoma cells isolated directly from pleural effusion or
ascites fluids and tested in a three days survival assay in human
blood extract based medium as shown in FIG. 2. (The 5 step
intensity scale of the heat map corresponds to 50% survival rate at
the given drug dilution. Black: more than 50% survival at the
highest drug concentration, highest intensity red less than 50%
survival at the highest drug dilution).
[0054] FIG. 4: Drug sensitivity pattern of 42 different primary
Acute Myeloid Leukemia cells isolated directly from the peripheral
blood of patients and tested in a three days survival assay in
human blood extract based medium as shown in FIG. 2. (The 5 step
intensity scale of the heat map corresponds to 50% survival rate at
the given drug dilution. Black: more than 50% survival at the
highest drug concentration, highest intensity red less than 50%
survival at the highest drug dilution).
[0055] FIG. 5: Drug sensitivity pattern of 74 different primary
Chronic Lymphoid Leukemia cells isolated directly from the
peripheral blood of patients and tested in a three days survival
assay in human blood extract based medium as shown in FIG. 2. (The
5 step intensity scale of the heat map corresponds to 50% survival
rate at the given drug dilution. Black: more than 50% survival at
the highest drug concentration, highest intensity red less than 50%
survival at the highest drug dilution).
[0056] FIG. 6: Drug sensitivity pattern of 216 human primary tumor
cells tested in a three days survival assay in human blood extract
based medium as shown in FIG. 2. (The 5 step intensity scale of the
heat map corresponds to 50% survival rate at the given drug
dilution. Black: more than 50% survival at the highest drug
concentration, highest intensity red less than 50% survival at the
highest drug dilution).
[0057] FIG. 7: Drug sensitivity pattern of highly resistant primary
ovarium carcinoma cells that show significant sensitivity only for
two anti cancer drugs out of 29 tested. Our blood extract based
automated short term drug sensitivity assay allows the
identification of potentially effective drugs even in apparently
hopeless cases of late stage cancer.
[0058] FIG. 8: The figure shows the result of a three days cell
survival assay. The tumor cells were isolated from an acute myeloid
leukemia (AML) patient and treated with different drugs, at
different concentrations in blood extract based medium or in a
traditional, rich cell culture medium--IMDM. The number of
surviving cells at the end of the incubation is plotted against the
relative drug concentration represented here as the proportion of
in vivo/in vitro Area Under Curve* values. The value pAUC=1
represents drug concentration regularly achieved in the serum of a
patient during chemotherapy. *AUC, Area Under Curve is a time
dependent integration of the drug concentration curve expressed as
(microgram/ml)*hours
[0059] FIG. 9: Similar drug sensitivity profile of primary human
carcinoma cells in blood extract based cell culture medium produced
from human, bovine or porcine blood. Three days survival assay as
described in FIG. 2.
EXAMPLE 1
Production of Total Human Blood Extract
[0060] Human blood from a healthy donor is obtained by
venopuncture. Alternatively animal blood (preferentially bovine or
porcine origin) is freshly collected under sterile conditions
during the time of slaughter. Coagulation is prevented by adding
100 IE Heparin per ml to the collecting bag. Subsequently the red
blood cells are lysed in order to release the intracellular content
of the red blood cells into the plasma. In an optimal embodiment of
the formulation the lysis is carried out by freezing the
anti-coagulated blood to -20.degree. C. in 50 ml aliquots, in
controlled temperature freezing device to achieve the optimal and
even disruption of red blood cell membranes by the help of
microscopic ice crystal formation. Subsequently the frozen blood is
stored at -80.degree. C. until further processing. To correct the
ion content changes, resulted by the hemolysis, and to supply small
molecular weight nutrients, the lysed blood is dialysed against
excess volume of cell culture medium such as Dulbeccos modified
Eagle medium (DMEM--D6546, Sigma-Aldrich Company) or Iscoves
modified DMEM (IMDM). In an optimal embodiment of the formulation
the 50 ml frozen blood, kept in Falcon plastic tube, is thawed in a
37.degree. C. water bath. The thawed blood lysate is filled into a
rehydrated Cellu SepH1 dialysis bag (0310-46, M.S.E. Micron
Separation Europe, Belgium--size exclusion limit 3500D). The bag is
closed under mild (1.2-1.4 bar) extra pressure over the normal
atmospheric pressure to remove air bubbles and to prevent further
volume expansion. The dialysis is carried out in the dark, for 24
hours, at 4.degree. C. against twenty fold excess fluid under
constant magnetic stirring. The red blood cell membrane ghosts are
removed by centrifugation. In an optimal embodiment of the
formulation the centrifugation is carried out a through a
microporous silica matrix equilibrated with the medium that was
used for the dialysis. The proper removal of the ghosts is
monitored and documented by taking 10 microliter samples from every
50 ml filtered lysates and checked using automated digital phase
contrast microscopy. In order to create a proper redox milieu, the
particle free blood lysate is supplemented with an extra reducing
agent. In an optimal embodiment of the formulation the agent is 0.5
mM reduced glutathione (G6013, Sigma-Aldrich Company). Subsequently
the lysate is vacuum filtered through a nitrocellulose membrane
(Corning 431153, Corning Inc.) with 0.22 micrometer pore size and,
immediately after the last filtration step flash frozen in liquid
nitrogen, in 3 ml aliquots. The frozen aliquots are stored at -80 C
until use in the biological assays. See FIG. 1 for the schematic
overview of the procedure.
EXAMPLE 2
Viability Assay of Drug Treated Primary Human Tumor Cells
[0061] Total blood lysate based cell culture medium is particularly
suitable for testing the drug sensitivity of primary human cells
that grow in suspension form, using short term in vitro survival
assays. For this assays the cells are purified from peripheral
blood, bone marrow or body cavity (peritoneal or pleural) effusions
using Ficoll gradient centrifugation. In the regular setup of the
assay, ten ml body fluid is treated with 100 IE/ml heparin in order
to prevent coagulation. The fluid is overlayed on 5 ml LymphoprepR
(Frezenius Kabi Norge AS) cushion and centrifuged for 10 minutes at
2500 rpm.
[0062] The cells from the gradient interphase are collected and
washed with pre-warmed cell culture medium containing 20% human
serum. The quality and tumor cell content of the sample is checked
and documented using phase contrast microscopy. If the tumor cell
count is below 70% the cells are further purified by negative
selection using an appropriate Dynabead cocktails reacting with the
contaminating normal (e.g. inflammatory) cells. The identity of the
tumor cells is verified in independent assays using
immunohistochemical markers. For example carcinoma cells in body
cavity effusions are identified on Cytospin slides with
immunofluorescence staining against pan-cytokeratin antibodies. The
presence of T, B, plasma-, NK cells, granulocytes and
macrophages/monocytes is routinely tested using for example anti
CD3, CD4, CD8, CD19, Ig, CD56, Mac1 antibodies. The drug
sensitivity of the tumor cells are tested on 384 well plates.
[0063] The pre-printed 384-well drug plates are produced using a
Biomek 2000 laboratory robot equipped with flat end stainless steal
metal pins with 50 nanoliter replica volumes. The master-plates are
created by serially diluting the drug stock solutions using
combination of single tip and 8 tip mode of the robot. In a typical
set-up we handle 30 different drugs in 4 different dilution series
(regularly 1.times. 5.times. 25.times. 125.times. diluted from the
stock, in 50% DMSO/PBS) in triplicates along with 24 control wells.
An example of a drug series with the highest employed drug
concentrations is shown in the table on FIG. 2. The drug
concentrations are selected considering the pharmacokinetic AUC
values for human subjects as established in clinical studies [14,
15]. When it is necessary, active metabolites are used instead of
the original inactive pro-drug (e.g. 4-OH cyclophosphamide).
[0064] For the short-term survival assay
2.times.10.sup.5-2.times.10.sup.6 tumor cells (approx. 500-4000
cells per well) are diluted in 12 ml freshly thawed total blood
lysate based cell culture medium. Thirty microliter samples are
distributed in the wells of the preprinted 384 well drug plate. The
cells are incubated for 72 hours in an incubator at 37.degree. C.
in 5% CO.sub.2 atmosphere. At the end of the incubation the cells
are differentially stained with fluorescence stain mixtures that
discriminate between live and dead cells (e.g. VitalDye-Biomarker,
Hungary) (FIG. 2). The content of the individual wells are
photographed using an automated inverted fluorescence microscope at
two different excitation wavelengths [16]. The captured pictures
are analyzed using digital image quantitation programs that count
number of living and dead cells from binary thresholded images on a
single cell level. The viability score after 3 days of incubation
with the indicated drug dilution was charted as heat maps created
by pairwise hierarchical clustering for different primary tumors
such as lung, ovarium and breast carcinomas (FIG. 3), acute myeloid
leukemias (FIG. 4), chronic lymphoid leukemias (FIG. 5). The
summary drug sensitivity map of 216 primary tumors created as
self-organizing map is shown in FIG. 6. These data clearly indicate
that even very resistant tumors (e.g. late stage ovarium carcinoma
ascites cells as shown in FIG. 7) can be sensitive for at least for
a few drugs when tested in our blood extract based cell culture
medium.
[0065] Total fluorescence intensity, cell size, cell surface
circumference as well as morphological parameters such as
circularity, feret diameter are recorded for subsequent High
Content Analysis (HCA). If cell cycle distribution measurements are
required the cells are subjected to a combination of
permeabilization and fixation treatment, such as detergent
treatment combined with aldehyde crosslinking. In practice, a
mixture of formaldehyde and Nonidet P-40 is added to the
fluorescently stained drug plates that yield 1 and 0.1%
concentration respectively. The plates are imaged again using the
parameters that were previously employed for dead cell recording.
The fluorescent dye that in the first round detected the dead cells
on the basis of staining the DNA in cells with compromised membrane
permeability is used in the second round to measure the total DNA
content in all the cells. Combination of cell cycle distribution,
cell viability and detailed cell morphology data provide a valuable
set of information to characterize the effect of a particular drug
on the primary tumor cells. For example cell cycle stop, or the
rounding up of adherent cells, provide alternative, more-subtle,
toxicity endpoints on a tumor cell population where simple
viability assay would show no difference.
EXAMPLE 3
Comparison with Classic Cell Culture Media (Decreased Drug
Sensitivity)
[0066] Incubation of primary human carcinoma and leukemia cells in
blood extract based and conventional medium consistently yielded
higher overall survival rate and decreased sensitivity to the
cytotoxic effects of different anti-cancer drugs. (FIG. 8)
EXAMPLE 4
Comparison of Blood Extracts of Human, Bovine and Porcine
Origin
[0067] Blood extract were generated from freshly collected blood
from newly slaughtered pigs or calves. The blood was collected
under sterile conditions and processed as described under Example
1.
[0068] Parallel assays using freshly isolated primary human
carcinoma cells showed comparable drug sensitivity patterns.
Primary human leukemia cells showed somewhat better survival in
human blood extracts for some of the drugs. These data suggest that
high throughput drug screening assays on primary human carcinoma
cells form ascites or pleural effusion may be efficiently carried
out using blood extract of animal origin whereas testing of drug
sensitivity pattern of cells from human leukemia patients should be
carried out in blood extract of human origin. (FIG. 9).
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