U.S. patent application number 10/208480 was filed with the patent office on 2004-02-05 for method for preparing cell cultures from biological specimens for chemotherapeutic and other assays.
This patent application is currently assigned to Precision Therapeutics, Inc.. Invention is credited to Gimigliano, Sheri, Kornblith, Paul L..
Application Number | 20040023375 10/208480 |
Document ID | / |
Family ID | 31186831 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023375 |
Kind Code |
A1 |
Kornblith, Paul L. ; et
al. |
February 5, 2004 |
Method for preparing cell cultures from biological specimens for
chemotherapeutic and other assays
Abstract
An improved method for preparing a cell culture is disclosed.
The method includes culturing a multicellular tissue explant in the
presence of growth medium that is substantially free of enzymes
capable of digesting the explant and, subsequently, removing the
explant at a predetermined time.
Inventors: |
Kornblith, Paul L.;
(Pittsburgh, PA) ; Gimigliano, Sheri; (Pittsburgh,
PA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Assignee: |
Precision Therapeutics,
Inc.
Pittsburgh
PA
|
Family ID: |
31186831 |
Appl. No.: |
10/208480 |
Filed: |
July 30, 2002 |
Current U.S.
Class: |
435/366 ;
435/7.23 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 2800/52 20130101; C12N 2503/02 20130101; C12N 5/0693
20130101 |
Class at
Publication: |
435/366 ;
435/7.23 |
International
Class: |
G01N 033/574; C12N
005/08 |
Claims
What is claimed is:
1. A method for preparing a cell culture, the method comprising:
culturing a multicellular tissue explant in the presence of growth
medium that is substantially free of enzymes capable of digesting
said explant; and removing said multicellular tissue explant from
said growth medium at a predetermined time, thereby to produce a
cell culture monolayer.
2. The method of claim 1, further comprising preparing a cell
suspension from said cell culture monolayer.
3. The method of claim 1, further comprising inoculating cells from
said cell culture monolayer into at least one segregated site.
4. The method of claim 1, wherein said tissue explant comprises
tumor tissue.
5. The method of claim 1, further comprising the step of
mechanically fragmenting the multicellular tissue explant in a
medium that is free of enzymes that are capable of digesting said
explant.
6. The method of claim 1, comprising the step of removing said
multicellular tissue explant from said growth medium prior to an
emergence of a substantial number of stromal cells from said
explant, thereby to produce a cell culture monolayer that is
predominantly composed of non-stromal cells.
7. The method of claim 6 wherein said non-stromal cells comprise
tumor cells.
8. The method of claim 1, wherein the cell culture monolayer
exhibits a percent confluency, and wherein the step of removing
said multicellular tissue explant comprises removing said
multicellular tissue explant at about 10 to about 50 percent
confluency.
9. The method of claim 8, wherein the step of removing said
multicellular tissue explant comprises removing said multicellular
tissue explant at about 15 to about 25 percent confluency.
10. The method of claim 8, wherein the step of removing said
multicellular tissue explant comprises removing said multicellular
tissue explant at about 20 percent confluency.
11. A cell culture preparation resulting from the method of claim 1
or 10.
12. A method for determining the chemosensitivity of a tissue in a
patient to an agent, the method comprising the steps of: (a)
culturing a multicellular tissue explant from a patient in the
presence of growth medium that is substantially free of enzymes
capable of digesting said explant so as to produce a cell culture
monolayer; (b) removing said multicellular tissue explant from said
growth medium at a predetermined time; (d) inoculating cells from
said cell culture monolayer into at least one segregated site; (e)
exposing said segregated site to at least one agent; and (f)
assessing the chemosensitivity of said cells in said segregated
site, wherein said chemosensitivity of said cells is indicative of
the chemosensitivity of the tissue in the patient.
13. The method of claim 12, wherein said multicellular tissue
explant comprises tumor tissue.
14. The method of claim 13, wherein the cell culture monolayer
exhibits a percent confluency, and wherein the step of removing
said multicellular tissue explant comprises removing said
multicellular tissue explant at about 10 to about 50 percent
confluency.
15. The method of claim 14, wherein the step of removing said
multicellular tissue explant comprises removing said multicellular
tissue explant at about 15 to about 25 percent confluency.
16. The method of claim 14, wherein the step of removing said
multicellular tissue explant comprises removing said multicellular
tissue explant at about 20 percent confluency.
17. A method for determining the efficacy of an agent, the method
comprising the steps of: (a) preparing a cell culture in accordance
with claim 3; (b) exposing said segregated site to at least one
agent; and (c) assessing the chemosensitivity of said cells in said
segregated site, thereby to determine the efficacy of said
agent.
18. The method of claim 17, wherein the cell culture monolayer
exhibits a percent confluency, and wherein the step of removing
said multicellular tissue explant comprises removing said
multicellular tissue explant at about 15 to about 25 percent
confluency.
19. The method of claim 18, wherein the step of removing said
multicellular tissue explant comprises removing said multicellular
tissue explant at about 20 percent confluency.
20. A method for identifying an agent having anti-tumorogenic
effect, the method comprising the steps of: (a) preparing a cell
culture in accordance with claim 4; (b) inoculating cells from said
cell culture monolayer into a plurality of segregated sites (c)
exposing each of said segregated sites to an agent; and (d)
assessing the chemosensitivity of said cells in each of said
segregated sites, thereby to identify an agent having an
anti-tumorogenic effect.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] Reference is made to U.S. Ser. No. 09/040,161, filed Mar.
17, 1998, which is a continuation of U.S. Ser. No. 08/679,056,
filed Jul. 12, 1996, now U.S. Pat. No. 5,728,541. The disclosure of
each of the foregoing of which is incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods for the preparation of a
cell culture monolayer, and more particularly to methods for the
preparation of a tumor cell culture monolayer that substantially
comprises tumor cells.
BACKGROUND
[0003] Prior to approval for medical use in the United States, all
pharmaceutical agents are subjected to rigorous testing for
efficacy and safety. Typically, methods of assessing the efficacy
of a pharmaceutical agent include complex studies of pooled patient
samples or pooled data, and statistical interpretation of the
results. The conclusions that follow such studies are inherently
generalized or averaged over the subject patient population. With
pharmaceutical agents, however, and particularly with cancer
chemotherapeutic agents, the efficacy of an agent in treating an
individual patient can vary greatly from the generalized data,
often to the detriment of the individual patient. The need has long
been recognized for a method of assessing the therapeutic potential
of pharmaceutical agents, including but not limited to
chemotherapeutic agents, for their specific efficacy in an
individual patient.
[0004] Assays exist which expose malignant tissue of various types
to pharmaceutically-active agents for the purpose of assessing the
best choice for therapeutic administration. For example, in
Kruczynski, A., et al., "Evidence of a direct relationship between
the increase in the in vitro passage number of human non-small-cell
lung cancer primocultures and their chemosensitivity," Anticancer
Research, vol. 13, no. 2, pp. 507-513 (1993), chemosensitivity of
non-small-cell lung cancers was investigated in in vivo grafts, in
in vitro primocultures, and in commercially available cancer cell
lines. The increase in chemosensitivity was documented and
correlated with morphological changes in the cells in question.
Often, animal model malignant cells and/or established cell
cultures are tested with prospective therapy agents, see for
example Arnold, J. T., "Evaluation of chemopreventive agents in
different mechanistic classes using a rat tracheal epithelial cell
culture transformation assay," Cancer Res., vol. 55, no. 3, pp.
537-543 (1995).
[0005] According to prior art methods of using specific patient
tumor cells to form an in vitro assay particular to that patient,
the cells are harvested (biopsied) and trypsinized (connective
tissue digested with the enzyme trypsin) to yield a cell suspension
suitable for conversion to the desired tissue culture form. The in
vitro tissue culture cell preparations which result from these
prior art methods typically fail to accurately replicate the
chemosensitivity of the original tumor or other cell biopsy. This
inability arises, in part, because the heterogeneity of cell
population in the tumor tissue has been disturbed in culture, or
entirely obliterated such that the cell culture preparation is
essentially monoclonal. Moreover, prior art methods of culturing
actual patient tissue samples inevitably result in cell cultures
with a significant level of nontarget cells such as fibroblasts or
other stromal cells, which have a tendency to outgrow the target
tumor cells in a cell culture. Furthermore, standard cloning and
tissue culture techniques are complicated and expensive for use in
individualized patient testing. Thus, a need remains for improved
and efficient methods of cell culture preparation that provide a
heterogeneous cell population that substantially comprises target
cells from a particular patient. Such a cell culture preparation is
useful in drug or chemotherapeutic agent screening to provide
information indicative of the in vivo reactivity of the cells, and
thus the specific efficacy as to a particular patient.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods for preparing a cell
culture from a multicellular tissue extract. Cell cultures of the
invention have the advantage of closely resembling the in vivo cell
population from which they were obtained, thus providing an
accurate and reliable proxy for the cell population in vivo. For
example, a tumor cell culture of the invention comprises a
population of cells that mimics the tumor cell population in the
patient from whom a tissue explant is obtained. This allows
chemosensitivity and chemoresistivity testing that is
highly-reliable in predicting the effects of therapeutic agents on
the tumor in vivo. The invention is based, in part, on the insight
that timely removal of a cellular explant from culture results in a
culture that is highly indicative of in vivo cell population. The
invention provides further benefits recognized by culturing tissue
explants in a growth medium that is essentially free of digestive
enzymes.
[0007] Accordingly, in one aspect, the invention provides a cell
culture system in which a multicellular tissue explant is placed in
a growth medium and is removed from the growth medium at a
predetermined time. The explant is removed prior to the emergence
from the explant of a substantial number of non-target cells,
resulting in a monolayer of cells that is enriched for the cell
population of interest. For example, it has been discovered that
cells emerge as a monolayer from a cultured tumor tissue explant in
an orderly fashion, the tumor cells emerging first, followed by
stromal cell populations. If the tumor cell explant remains in
culture, the stromal cells have been found to dominate the tumor
cells in culture. This creates a culture that is enriched from
non-target stromal cells and that is not reflective of the in vivo
cell population. Thus, in a tumor cell culture, the explant is
removed from the growth medium prior to the emergence of a
substantial number of stromal cells from the explant. This provides
a cell culture monolayer that is predominantly composed of tumor
cells. It has also been discovered that the cell cultures described
above produce optimal results in cell culture medium that is
essentially free of digestive enzymes.
[0008] The time at which an explant is removed from its culture
medium depends upon the type of cells being cultured, the rate of
emergence of various cell types, and the desired purity of the
resulting cell culture monolayer. This can be determined
empirically for a given cell type. In the case of tumor cells, the
multicellular tissue explant is preferably removed when the cell
culture monolayer is at about 10 to about 50 percent confluency. In
a preferred embodiment, the multicellular tissue explant is removed
at about 15 to about 25 percent confluency. In a particularly
preferred embodiment, the explant is removed at about 20 percent
confluency.
[0009] The invention further comprises the preparation of a cell
suspension from the cell culture monolayer. A tissue explant is
cultured in an appropriate medium and is removed at a predetermined
time, resulting in a monolayer enriched for the cells of interest.
A suspension is then made from the monolayer and cells of the
suspension are inoculated into at least one segregated site. In one
embodiment, a chemosensitivity assay is performed on the inoculated
cell suspension by exposing the segregated site to at least one
agent and assessing the chemosensitivity of the cells in the
segregated site. Chemoresistivity assays are similarly
performed.
[0010] In another embodiment, the invention provides methods for
determining the chemosensitivity of a tissue in a patient by
determining the chemosensitivity of a cell culture preparation from
the patient. In yet another embodiment, the invention provides
methods for identifying an agent having anti-tumorogenic effect in
a patient by assessing the chemosensitivity of segregated sites of
cells from a tumor cell culture prepared according to the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention provides methods for preparing a cell
culture monolayer by culturing a tissue sample from a patient.
Ultimately, the culture may be used to screen at least one
candidate therapeutic or chemotherapeutic agent for efficacy as to
a specific patient, in which a tissue sample from the patient is
harvested and separately exposed to a plurality of treatments
and/or therapeutic agents for the purpose of objectively
identifying the chemosensitivity or chemoresistivity of the tissue
sample and the best treatment or agent for the patient. Tissue
sample preparation techniques render this method practically as
well as theoretically useful. According to the invention, the
initial cohesive multicellular particulates (explants) of the
tissue sample are prepared mechanically, rather than enzymatically,
for initial tissue culture monolayer preparation. The multicellular
tissue explant is removed from the culture growth medium at a
predetermined time to both allow for the growth of target cells and
prevent substantial growth of non-target cells such as fibroblasts
or stromal cells.
[0012] An important application of the present invention is the
screening of chemotherapeutic agents and other antineoplastic or
anti-tumorogenic therapies against tissue culture preparations of
tumorogenic cells from the patient from whom the sample is
biopsied. Related anti-cancer therapies which can be screened using
the methods of the invention are both radiation therapy and agents
which enhance the cytotoxicity of radiation, as well as
immunotherapeutic anti-cancer agents. Screening processes for
treatment or therapeutic agents for nonmalignant syndromes are also
embraced within this invention, however, and include without
limitation, agents which combat hyperproliferative diseases, such
as psoriasis, or wound healing agents. Nor is the present efficacy
assay limited only to the screening of active agents which speed up
(healing) or slow down (anti-cancer, anti-hyperproliferative) cell
growth because agents intended to enhance or to subdue
intracellular biochemical functions may be tested in the present
tissue culture system as well. For example, the formation or
blocking of enzymes, neurotransmitters and other biochemicals may
be screened with the present assay methods prior to treatment of
the patient.
[0013] By way of example, in one embodiment of the invention, a
cell culture monolayer in accordance with the invention is prepared
using the following procedure. Many aspects of the following
procedure may be altered as necessary and as well known in the art.
A biopsy of normecrotic, non-contaminated tissue is harvested from
the patient by any suitable biopsy or surgical procedure known in
the art. In a preferred embodiment, the tissue sample is tumor
tissue. In one embodiment, the biopsy is at least about 100 mg.
Biopsy sample preparation generally proceeds under sterile
conditions. Cohesive multicellular particulates (explants) are
prepared from the tissue sample using mechanical fragmentation. In
one embodiment, this mechanical fragmentation of the explant occurs
in a medium substantially free of enzymes that are capable of
digesting the explant. In a preferred embodiment, the tissue sample
is minced with sterile scissors to prepare the explants. In a
particularly preferred embodiment, the tissue sample is
systematically minced by using two sterile scalpels in a
scissor-like motion, or mechanically equivalent manual or automated
opposing incisor blades. This cross-cutting motion creates smooth
cut edges on the resulting tissue multicellular particulates. In
one embodiment, multicellular particulates measuring about 1
mm.sup.3 may be produced. After the tissue sample has been minced,
the particles are plated in culture flasks (for example, 9 explants
per T-25 or 20 particulates per T-75 flask). The explants may be
evenly distributed across the bottom surface of the flask, followed
by initial inversion for about 10-15 minutes. The flask may then be
placed in a non-inverted position in a 37.degree. C. CO.sub.2
incubator for about 5-10 minutes. In another embodiment in which
the tissue sample comprises brain cells, the flasks are placed in a
35.degree. C., non-CO.sub.2 incubator. Flasks should be checked
regularly for growth and contamination. According to a preferred
embodiment of the invention, the multicellular explant is removed
from the cell culture at a predetermined time, as described below.
Over a period of a few weeks a monolayer will be produced. With
respect to the culturing of tumor cells, it is believed (without
any intention of being bound by the theory) that tumor cells grow
out from the multicellular explant prior to stromal cells. Thus, by
initially maintaining the tissue cells within the explant and
removing the explant at a predetermined time, growth of the tumor
cells (as opposed to stromal cells) into a monolayer is
facilitated.
[0014] The use of the above procedure to form a cell culture
monolayer culture maximizes the growth of tumor cells from the
tissue sample, and thus optimizes ensuing tissue culture assay of
various agents (e.g., chemotherapeutic agents) to be tested. Once a
primary culture and its derived secondary monolayer tissue culture
has been initiated, the growth of the cells may be monitored to
oversee growth of the monolayer and ascertain the time to initiate
the chemotherapy assay and to determine the growth rate of the
cultured cells. Prior to the chemotherapy assay, monitoring of the
growth of cells may be conducted by visual monitoring of the flasks
on a periodic basis, without killing or staining the cells and
without removing any cells from the culture flask. Data from
periodic counting is then used to determine growth rates which may
or may not be considered parallel to growth rates of the same cells
in vivo in the patient. If growth rate cycles can be documented,
for example, then dosing of certain active agents can be customized
for the patient. The same growth rate can be used to evaluate
radiation treatment periodicity, as well. It should be noted that
with the growth rate determinations conducted while the monolayers
grow in their flasks, the present method requires no hemocytometry,
flow cytometry or use of microscope slides and staining, with all
their concomitant labor and cost.
[0015] Monolayer growth rate may be monitored using, for example, a
phase-contrast inverted microscope. In one embodiment, culture
flasks are incubated in a (5% CO.sub.2) incubator at about
37.degree. C. The flask is placed under the phase-contrast inverted
microscope, and ten fields (areas on a grid inherent to the flask)
are examined using the 10.times. objective. In general, the ten
fields should be non-contiguous, or significantly removed from one
another, so that the ten fields are a representative sampling of
the whole flask. Percentage cell occupancy for each field examined
is noted, and averaging of these percentages then provides an
estimate of overall percent confluency in the cell culture. When
patient samples have been divided between two or among three or
more flasks, an average cell count for the total patient sample
should be calculated. The calculated average percent confluency
should be entered into a process log to enable compilation of
data--and plotting of growth curves--over time. Alternatively,
confluency may be judged independently for each flask. Monolayer
cultures may be photographed to document cell morphology and
culture growth patterns. The applicable formula is: 1 Percent
confluency = estimate of the area occupied by cells total area in
an observed field
[0016] Percent confluency=estimate of the area occupied by cells
total area in an observed field
[0017] As an example, therefore, if the estimate of area occupied
by the cells is 30% and the total area of the field is 100%,
percent confluency is 30/100, or 30%.
[0018] Following initial culturing of the multicellular tissue
explant, the tissue explant is removed from the growth medium at a
predetermined time. In one embodiment, the explant is removed from
the growth medium prior to the emergence of a substantial number of
stromal cells from the explant. Alternatively, the explant may be
removed according to the percent confluency of the cell culture. In
one embodiment of the invention, the explant is removed at about 10
to about 50 percent confluency. In a preferred embodiment of the
invention, the explant is removed at about 15 to about 25 percent
confluency. In a particularly preferred embodiment, the explant is
removed at about 20 percent confluency. By removing the explant in
either of the above manners, a cell culture monolayer predominantly
composed of target cells (e.g., tumor cells) is produced. In turn,
a substantial number of non-target cells, such as fibroblasts or
other stromal cells, fail to grow within the culture. Ultimately,
this method of culturing a multicellular tissue explant and
subsequently removing the explant at a predetermined time allows
for increased efficiency in both the preparation of cell cultures
and subsequent assays of various agents using the cultures.
Adaptation of the above protocol for non-tumor cells is
straightforward and generally constitutes an equivalent
procedure.
[0019] The essence of the invention thus includes the important
feature of the simplicity of the present system--cohesive
multicellular explants of the patient tissue to be tested are used
to form cell monolayers; growth of those monolayers may be
monitored for accurate prediction of correlating growth of the same
cells in vivo; explants are removed from the growth medium at a
predetermined time, and differing concentrations of a number of
active agents may be tested for the purpose of determining
chemosensitivity of the tissue sample and the most appropriate
agent and concentration of that agent for actual patient exposure
(according to the calculated cell growth rates). It is also
important to note, in the context of the invention, that the
present system allows in vitro tests to be conducted in suspensions
of tissue culture monolayers grown in nutrient medium under fast
conditions (a matter of weeks), rather than with single cell
progeny produced by dilution cloning over long periods of time. In
some cases, the present invention provides a cell culture for a two
stage assay for both cytotoxicity and the longer-term growth
inhibitory.
EXAMPLE
Chemosensitivity Assay
[0020] Methods of the invention include methods for determining the
efficacy of an active agent. The performance of the
chemosensitivity assay used for screening purposes depends on the
ability to deliver a reproducible cell number to each row in a
plate and/or a series of plates, as well as the ability to achieve
an even distribution of cells throughout a given well. The
following exemplary procedure assures that cells are reproducibly
transferred from flask to microtiter plates, and cells are evenly
distributed across the surface of each well.
[0021] An initial step in preparing the microtiter plates is
preparing and monitoring the monolayer as described above with the
removal of the explant at 20 percent confluency. The following
example shows an exemplary protocol which is susceptible of
variation as will be apparent to one skilled in the art. Cells were
removed from the culture flask and a cell pellet was prepared by
centrifugation. The cell pellet derived from the monolayer was then
suspended in 5 ml of the growth medium, mixed in a conical tube and
subsequently rocked back and forth 10 times. A 30 .mu.l droplet
from the center of the conical tube was pipetted into one well of a
96 well plate. A fresh pipette was then used to pipette a 30 .mu.l
aliquot of trypan blue solution, which was added to the same well,
and the two droplets were mixed with repeated pipette aspiration.
The resulting admixture was then applied to a hemocytometer chamber
for examination using a standard light microscope. Cells were
counted in all of four hemocytometer quadrants, under 10.times.
magnification. Only those cells which had not taken up the trypan
blue dye were counted. Using means known in the art, the quadrant
count values were checked, logged, multiplied by 10.sup.4 to give
cells/ml, and the total amount of fluid (growth medium) necessary
to suspend remaining cell aliquots was calculated accordingly.
[0022] After the desired concentration of cells in medium has been
determined, additional cell aliquots from the monolayer were
suspended in growth medium via rocking and then, loaded into a
Terasaki dispenser. Aliquots of the prepared cell suspension were
delivered into the microtiter plates using Terasaki dispenser
techniques known in the art. Alternatively, an electronic
multichannel pipettor commercially available from Matrix Technology
Corp. may be used. A plurality of plates may be prepared from a
single cell suspension as needed. Plates were subsequently
incubated in an incubator box by means known in the art. Upon
preparation of the cell suspension, cells from the suspension may
be inoculated into segregated sites for subsequent assays. At least
one agent may be exposed to the segregated sites to determine the
chemosensitivity of the tissue samples, as well as the therapeutic
or chemotherapeutic effects of the agents on the tissue sample.
[0023] The following example provides an exemplary protocol for
assaying active agents in accordance with the invention. During
this portion of the inventive assay, the appropriate amount of
specific active agent was transferred into the microtiter plates
prepared as described above. A general protocol, which may be
adapted, follows. Each microtiter plate was microscopically
examined for cell adhesion. Control solution was dispensed into
delineated rows of wells within the grid in the microtiter plate,
and appropriate aliquots of active agent to be tested were added to
the remaining wells in the remaining rows. Ordinarily, sequentially
increasing concentrations of the active agent being tested were
administered into progressively higher numbered rows in the plate.
The plates were then incubated in an incubator box at 37.degree. C.
under 5% CO.sub.2. After a predefined exposure time, the plates
were blotted with sterile gauze to remove the agent, washed with
Hank's Balance Salt Solution, flooded with growth medium, and
replaced in the incubator in an incubator box for a predefined time
period, after which the plates were fixed and stained for
evaluation.
[0024] Fixing and staining may be conducted according to a number
of suitable procedures; the following is representative. After
removal of the plates from the incubator box, culture medium were
poured off and the plates were flooded with Hank's Balance Salt
Solution. After repeated flooding (with agitation each time) the
plates were then flooded with reagent grade ethanol for 2-5
minutes. The ethanol was then poured off. Staining was accomplished
using a DAPI (4', 6-diamidino-2-phenylindole, dilactate) staining
method. Each plate was flooded with a DAPI/water solution, with a
concentration of about 400 nM, and allowed to stand for at least 10
minutes, after which the DAPI/water was poured into a beaker. The
plates were then dipped into a beaker of running water to remove
the excess DAPI. Cells per well may then be counted manually or by
automated and/or computerized means, to derive data regarding
chemosensitivity of cells at various concentrations of exposure.
One particularly useful computer operating environment for counting
cells is the commercially available Zeiss Axiovert S100 Automatic
Inverted Flourescence Microscope and Computer.
[0025] The above procedures do not change appreciably when cell
growth promoters are assayed rather than cell arresting agents such
as chemotherapeutic agents. The present assay allows cell death or
cell growth to be monitored with equal ease. In any case,
optimization of use of the present system will involve the
comparative testing of a variety of candidate active agents, for
selection of the best candidate for patient treatment based upon
the in vitro results. One particularly advantageous embodiment of
the above-described invention comprises a two-stage assay for
cytotoxicity followed by evaluation of longer-term inhibitory
effect chemotherapeutic agents may thus be evaluated separately for
both their direct chemotherapeutic effect as well as for their
longer duration efficacy.
[0026] The invention is not to be limited only to the illustrative
description provided herein. Variations, modifications, and other
implementations of what is described herein will occur to those of
ordinary skill without departing from the spirit and scope of the
invention.
* * * * *