Method for conducting the cytotoxicity assays on tumor cells

A method and apparatus for assaying the sensitivity of biopsied tumor cells to chemotherapeutic agents using a predetermined amount of chemotherapeutic agent in an easily deliverable form is disclosed and claimed.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
this invention relates to a method and apparatus for conducting 
cytotoxicity assays on human biopsied tumor cells. 
2. Description of the Prior Art 
In 1955, Karnofsky presented to an audience of skeptics the ability of 
alkylating agents and antimetabolites to exhibit some chemotherapeutic 
activity in a limited number of human tumors. Tumor chemotherapy has 
greatly advanced since then and maintains an important role in the 
treatment of many tumors. Currently, chemotherapy can cure more than 16 
tumor types, including hematological neoplasms; sarcomas; testicular, 
gestational, trophoblastic, and Wilm's tumors; and small cell lung and 
ovarian cancers. Other tumors curable in the adjuvant setting are breast 
and colon cancers. Advances in chemotherapeutics are ongoing: more 
effective drugs, drug analogs with less toxicity, and drugs modified for 
improved tissue uptake and longer plasma half life are being developed. 
DeVita, V.T. Jr, Cancer Principles and Practice of Oncology, Principles of 
Chemotherapy (V.T. DeVita, Jr., S. Hellman, S.A. Rosenberg eds.), J.B. 
Lippincott Co., Philadelphia, Pa., 1989. 
Currently, tumor chemotherapeutic treatment is based upon standard 
practices resulting from empirical drug selection or established 
protocols. Von Hoff, D.D., L. Weisenthal, In Vitro Methods to Predict for 
Patient Response to Chemotherapy, Advances in Pharmacology and 
Chemotherapy, 17:133-156, 1980. Woltering, Eugene A., Administration of 
Cytotoxic Chemotherapeutic Agents Without Predictive Information, 
Laboratory of Medicine, 21:82, 1990. Variations in patient response to 
standard therapy are often the result of the highly heterogeneous nature 
of human tumors, both among different tumor types and within the same 
tumor type in an individual patient. This heterogeneity is reflected in 
the chemosensitivity of malignant cells. Variations in response can render 
unattainable the primary goals of chemotherapy--to maximize the effects of 
therapy on the tumor and to prevent side effects of therapy for the 
patient. Chemotherapy can offer the best chance of survival but it also 
has adverse effects that can be devastating, such as patient toxicity, 
immune system suppression, loss of time due to an ineffective treatment 
regimen, and the development of drug resistance. 
A predictive assay is particularly important for cancer chemotherapy since 
is identifies ineffective drugs whose side effects are potentially 
life-threatening. The development of an in vitro assay which could predict 
the response of an individual's tumor cells to chemotherapeutics has been 
a longstanding objective in cancer research. The pioneers in this field 
include: Hamburger, Q.W., and S.E. Salmon, Primary Bioassay of Human Tumor 
Stem Cells, Science, 197:461-463, 1977, Hamburger, Anne W., The Human 
Tumor Clonogenic Assay as a Model System in Cell Biology, International 
Journal of Cell Cloning, 5:89-107, 1987, Scheithauer, W., G.M. Clark, S.E. 
Salmon, W. Dorda, R.HY. Shoemaker, D.D. Von Hoff, Model for Estimation of 
Clinically Achievable Plasma Concentrations for Investigational Anticancer 
Drugs in man, Cancer Treatment Reports, 70:1379, Shoemaker, R.H., M.K. 
Wolpert-DeFilippes, R.W. Makuch, Application of the Human Tumor Clonogenic 
Assay for New Drug Screening, Stem Cells, 1:308, 1981, Shoemaker, R.H., 
M.K. Wolpert-DeFilippes, R.W. Makuch, Use of the Human Tumor Clonogenic 
Assay for New Drug Screening, Proc. Amer. Assoc. Cancer Research, 24:1231, 
1983, Shoemaker, R.H., M.K. Wolpert-DeFilippes, J.M. Venditti, IV., Human 
Tumors in the Screening of Cytostatics, Behring Inst. Mitt., 74:262, 1984, 
Alberts, D.S., H.S.G. Chen, Cloning of Human Tumor Cells, (S.E. Salmon 
ed.), 351-359Alan R. Liss, Inc., New York, N.Y., 1980, Alberts, D.S., 
H.S.G. Chen, L. Young, T.E. Moon, S.A. Loesch, E.A. Surwit, S.E. Salmon, 
Improved Survival for Relapsing Ovarian Cancer (OVCA) Patients Using the 
Human Tumor Stem Cell Assay (HTSCA) to Select Chemotherapy, Proc. Am. 
Assoc. Cancer Research, 22:461, 1981, Alberts, D.S., S.E. Salmon, E.A. 
Surwit, H.S.G. Chen, T.E. Moon, L. Young, Combination Chemotherapy (CRx) 
In Vitro With the Human Tumor Stem Cell Assay (HTSCA), Cancer Chemother, 
Pharmcol., 6:253, 1981, Von Hoff, Daniel D., James Casper, Edward Bradley, 
John Sandbach, Donna Jones, Robert Makuch, Association Between Human Tumor 
Colony-Forming Assay Results and Response of an Individual Patient's Tumor 
to Chemotherapy, American Journal of Medicine, 70:1027-1032, 1981., Von 
Hoff, Daniel D., Gary M. Clark, Brian J. Stogdill, Michael F. Sarosdy, 
Michael T. O'Brien, James T. Casper, Douglas E. Mattox, Carey P. Page, 
Anatolio B. Cruz, and John F. Sandbach, Prospective Clinical Trial of a 
Human Tumor Cloning System, Cancer Research, 43:1926-1931, 1983, Hanauske, 
Axel-R., Daniel D. Von Hoff, Clinical Correlations with the Human Tumor 
Cloning Assay, Cancer Investigation, 3(6):541-551, 1985., Von Hoff, Daniel 
D., In Vitro Predictive Testing: The Sulfonamide Era, International 
Journal of Cell Cloning, 5:179-190, 1987, Von Hoff, Daniel D., Commentary, 
He's Not Going to Talk About In Vitro Predictive Assays Again, Is He?, 
Journal of the National Cancer Institute, 82:96-101, 1990. 
Current in vitro chemosensitivity methods include: clonging of human tumors 
on double layer soft agar, i.e. human tumor cloning assay (HTCA); subrenal 
capsule assay method, fluorescent cytoprint assay (Rotman RIVCA) and 
tritiated thymidine uptake assay. 
The traditional in vitro method of growing human tumor cells in semi-solid 
agar developed by Salmon and Hamburger is referred to as the human tumor 
clonging assay (HTCA). In this method, solid tumors or malignant fluid 
from cancer patients may be used as the tumor cell source. The tumor 
specimens are mechanically and enzymatically dissociated to fulfill the 
requirement of a single-cell suspension. When short-term drug treatment is 
being evaluated, the single-cell suspension is incubated in media with or 
without the therapeutic drug. After the cells are exposed to the drug for 
one hour, control and treated cells are plated on agar. For continuous 
drug treatment, the drug is added directly to the top agar layer 
containing cells in a 2-layer system. In both cases, the cells are 
incubated for 14-21 days and observed for colony formation. The difference 
in the number of colonies counted in plates containing drug treated cells 
and in control plates is used to determine drug responsiveness, Shoemaker, 
Robert H., Mary K. Wolpert-DeFilippes, David H. Kern, Michael M. Lieber, 
Robert W. Makuch, Jeannete R. Melnick, William T. Miller, Sydney E. 
Salmon, Richard M. Simon, John M. Venditti and Daniel D. Von Hoff, 
Application of a Human Tumor Colony Forming Assay to a New Drug 
Sensitivity, Cancer Research, 45:2145-2153, 1985, Woltering, Eugene A., 
Tumor Chemosensitivity Testing: An Evolving Technique, Laboratory 
Medicine, 2:82-84, 1990. 
The subrenal capsule assay method differs from the traditional HTCA by 
utilizing tumor fragments, measuring the responsiveness of multiple cell 
populations rather than single-cell suspensions. Subrenal capsule is an in 
vivo assay utilizing human tumor specimens as first-generation transplant 
xenografts in athymic mice. The predictability of drug resistance with the 
subrenal capsule assay has not been found to be superior to the HTCA. The 
greatest advantage is that more tumor specimens can be successfully grown 
using subrenal capsule than HTCA, Woltering, Eugene A., Tumor 
Chemosensitivity Testing: An Evolving Technique, Laboratory Medicine, 
2:82-84, 1990. However, due to the expense involved in maintaining mouse 
colonies and the technical expertise required, the subrenal capsule assay 
has not become a routine test. 
Rotman and coworkers have developed an in vitro chemosensitivity method 
(RIVCA) also referred to as the fluorescent cytoprint assay (FCA). In this 
assay, tumor fragments are exposed to the drug and cultured. The viability 
of the tumor cells is measured by their ability to hydrolyze fluoresce in 
diacetate and retain the fluorescein. The difference in the number of 
fluorescent fragments before and after drug treatment is used as a measure 
of drug response. RIVCA is not amenable to fluid specimens or to solid 
specimens yielding small cell aggregates, since only aggregates larger 
than 50-100 cells can be photographically recorded. The predictability of 
drug resistance with RIVCA is similar to that of HTCA, but a greater 
number of tumors can be grown in vitro and evaluated using RIVCA, 
Woltering, Eugene A., Tumor Chemosensitivity Testing: An Evolving 
Technique, Laboratory Medicine, 2:82-84, 1990. 
Due to difficulties in obtaining single-cell suspensions from solid tumors, 
long incubation times, and poor tumor growth in the HTCA, alternative 
methods have been evaluated. One of these methods uses the incorporation 
of a radionucleotide, such as tritiated thymidine, during DNA synthesis as 
an indication of cell viability and proliferation. Tumor preparations are 
exposed to drugs, either short-term or continuously, and cultured in 
liquid medium. Tritiated thymidine is added and the culture incubated for 
16-24 hours. Incorporated radionucleotides are harvested and counted with 
a scintillation counter. A decrease in the uptake of tritiated thymidine 
by tumor cells exposed to cancer chemotherapeutics indicates sensitivity 
of the tumor to the drug. This assay has several advantages over HCTA, 
SRA, and RIVCA: a shorter culture period (4-6 days) is required, a smaller 
sample size can be assayed, and the strict requirement for single-cell 
suspensions--a goal often unachievable for solid tumors--is eliminated. 
Another benefit of this assay is that the determination of tumor growth is 
quantitative and automated, in contrast to the subjective counting of 
colonies by a tissue culture technician. The clinical relevance of a 
radionucleotide detection system in an in vitro chemosensitivity assay has 
been documented: Kern, David H., Carol R. Drogemuller, Michale C. Kennedy, 
Susanne U. Hioldebrand-Zanki, Nobuhiko Tanigawa, and Vernon K. Sondak, 
Development of a Miniaturized, Improved Nucleic Acid Precursor 
Incorporation Assay for Chemosensitivity Testing of Human Solid Tumors, 
Cancer Research, 45;5435-5441, 1985; Daidone, Maria Grazia, Rosella 
Silvestrini, Ornella Sanfilippo, Nadia Zaffaroni, Marco Varini, Mario 
Delena, Reliability of an In Vitro Short-Term Assay to Predict the Drug 
Sensitivity of Human Breast Cancer, Cancer, 56:450-14 456, 1985, Tanigawa, 
Nobuhiko, David H. Kern, Yorinori Hikasa, and Donald L. Morton, Rapid 
Assay for Evaluating the Chemosensitivity of human Tumors in Soft Agar 
Culture, Cancer Research, 42:2159-2164, 1982, Wilson, A.P., C.H.J. Ford, 
C.E. Newman, A. Howell, A Comparison of Three Assays Used for the In Vitro 
Chemosensitivity Testing of Human Tumours, British Journal of Cancer, 
49:57-63, 1984. One group of investigators assaying cells from a variety 
of solid tumor types (breast, lung, and ovarian cancers, melanomas and 
sarcomas) found that 80% of the specimens were evaluable (280/351) with 
100% accuracy in predicting resistance and 50% accuracy in predicting 
sensitivity. Kern, David H., Carol R. Drogemuller, Michael C. Kennedy, 
Susanne U. Hildebrand-Zanki, Nobuhiko Tanigawa, and Vernon K. Sondak, 
Development of a Miniaturized, Improved Nucleic Acid Precursor 
Incorporation Assay for Chemosensitivity Testing of Human Solid Tumors, 
Cancer Research, 45:4335-5441, 1985. In another report, assaying only 
cells derived from breast cancers, the prediction of tumor sensitivity and 
resistance was 75% and 81% accurate, respectively. Daidone, Maria Grazia, 
Rosella Silvestrini, Ornella Sanfilippo, Nadia Zaffaroni, Marco Varini, 
Mario DeLena, Reliability of an In Vitro Short-Term Assay to Predict the 
Drug Sensitivity of Human Breast Cancer, Cancer, 56:450-456. 
In order to conduct tumor sensitivity assays the tumor must be maintained 
in culture. Epithelial cells are the select culture medium in this field. 
In a recently published report, Von Hoff, Daniel D., Commentary, He's Not 
Going to Talk About In Vitro Predictive Assays Again, Is He?, Journal of 
the National Cancer Institute, 82:96-101, 1990, based on nearly 14,000 
tumor samples, only 3,886 or 27.9% had sufficient in vitro growth for 
evaluation of drug sensitivity. Recent modifications of culture conditions 
and the development of sensitive detection methods have increased the 
capacity to obtain evaluable specimens. Hanauske, Axel-R., Daniel D. Von 
Hoff, Clinical Correlations with the Human Tumor Cloning Assay, Cancer 
Investigation, 3(6):541-551, 1985, Shoemaker, Robert H., Mary K. 
Wolpert-DeFilippes, David H. Kern, Michael M. Lieber, Robert W. Makuch, 
Jeannete R. Melnick, William T. Miller, Sydney E. Salmon, Richard M. 
Simon, John M. Venditti, and Daniel D. Von Hoff, Application of a Human 
Tumor Colony Forming Assay to New Drug Sensitivity, Cancer Research, 
45:2145-2153, 1985. The specific problem of inadequate in vitro tumor 
growth has been explored extensively. To achieve adequate tumor growth, a 
defined, selective medium is required that allows tumor cells, most 
commonly of epithelial origin, to actively proliferate while inhibiting 
the proliferation of normal cells, such as fibroblasts. It has become 
apparent, however, that "traditional" growth media and high serum 
concentrations are not optimal for epithelial tumor cell growth. Reid, 
Lola M., Generic Methods for Defined Hormonal and Matrix Conditions for 
Studies of Growth or Gene Expression in Differentiated Epithelia, Methods 
in Molecular Biology, (J.W. Pollard, J.M. Walker, eds.), Volume 5: Tissue 
Culture. Growth medium containing a high calcium (greater than 1 mM) and 
high serum (10-25%) concentration enhances the proliferation of 
fibroblasts. In contrast, epithelial cells exhibit the best growth in a 
low calcium (approximately 0.4 mM) and a low serum (1% and below) 
environment. Additionally, serum in the medium contributes to inconsistent 
results between assays due to lot-to-lot variations in the concentrations 
of several critical components in the serum. A low serum concentration in 
the medium reduces the impact of this variability. However, since 
epithelial tumor cells require specific growth factors and hormones which 
are present in the serum, reduction of the serum concentration 
necessitates the supplementation of those growth factors and hormones. 
Barnes, David and Gordon Satro, Methods for Growth of Cultured Cells in 
Serum-Free Medium, Analytical Biochemistry, 102:255-270, 1980. Therefore, 
a growth medium containing low calcium and serum concentrations 
supplemented with defined growth factors and hormones allows preferential 
growth of epithelial tumor cells, resulting in an increase in the number 
of evaluable specimens. Reid, Lola M., Generic Methods for Defined 
Hormonal and Matrix Conditions for Studies of Growth or Gene Expression in 
Differentiated Epithelia, Methods in Molecular Biology, (J.W. Pollard, 
J.M. Walker, eds.), Volume 5: Tissue Culture, Crickard, Kent, Ulla 
Crickard, Mahmood Yoonessi, Human Ovarian Carcinoma Cells Maintained on 
Extracellular Matrix Versis Plastic, Cancer Research, 43:2762-2767, 1983. 
Roswell Park Memorial Institute 1640 (RPMI) (Life Technologies, Grand 
Island, N.Y.) is a basal medium containing inorganic elements, energy 
sources, vitamins, amino acids, and a low concentration of calcium (0.67 
mM). RPMI, however, lacks the hormones and growth factors often necessary 
for proliferation of epithelial tumor cells. Various hormones and growth 
factors are typically added individually to the growth medium according to 
the requirements of the cell type being grown. Barnes, David and Gordon 
Sato, Methods for Growth of Cultured Cells in Serum-Free Medium, 
Analytical Biochemistry, 102:255-270, 1980, Ham, R.G., Importance of the 
Basal Nutrient Medium in the Design of Hormonally Defined Media, Cold 
Spring Harbor Laboratory, 9:39-60, 1982. 
In a review of 2300 patients, the correlation between an in vitro 
chemosensitivity assay and actual patient response indicated that the 
predictability of true sensitivity is 69% and true negative predictability 
91%. Scheithauer, W., G.M. Clark, S.E. Salmon, W. Dorda, R.H. Shoemaker, 
D.D. Von Hoff, Model for Estimation of Clinically Achievable Plasma 
Concentrations for Investigational Anticancer Drugs in Man, Cancer 
Treatment Reports, 70:1379, Von Hoff, Daniel D., James Casper, Edward 
Bradley, John Sandbach, Donna Jones, Robert Makuch, Association Between 
Human Tumor Colony-Forming Assay Results and Response of an Individual 
Patient's Tumor to Chemotherapy, American Journal of Medicine, 
70:1027-1032, 1981, Von Hoff, Daniel D., Gary M. Clark, Brian J. Stogdill, 
Michael P. Sarosdy, Michael T. O'Brien, James T. Casper, Douglas E. 
Mattox, Carey P. Page, Anatolio B. Cruz, and John F. Sandbach, Prospective 
Clinical Trial of a Human Tumor Cloning System, Cancer Research, 
43:1926-1931, 1983, Hanuske, Axel-R., Daniel D. Von Hoff, Clinical 
Correlations With the Human Tumor Cloning Assay, Cancer Investigation, 
3(6):541-551, 1985. These correlations have been the result of either 
retrospective or prospective single-arm studies. Only two prospective 
randomized trials have been performed. One prospective study was conducted 
with ovarian cancer patients in which the response rates, though not 
statistically significant, were 65% for the standard chemotherapy arm and 
85% for treatment based on in vitro assay results. Welander, C.E., T.M. 
Morgan, H.D. Homesley, Multiple Factors Predicting Responders to 
Combination Chemotherapy in Patients With Ovarian Cancer, In Human Tumor 
Cloning (S.E. Salmon, J.M. Trent eds.) Orland: Grune and Stratton, 
521-534, 1984. A larger scale prospective randomized trial was conducted 
on 133 advanced metastatic cancer patients. Patient response rates were 
21% for those who received single-agent chemotherapy based on in vitro 
assay results and only 3% in patients who received a clinician's choice of 
a single agent. Von Hoff, Daniel D., Commentary, He's Not Going to Talk 
About In Vitro Predictive Assays Again, Is He?, Journal of the National 
Cancer Institute, 82:96-101, 1990. These studies begin to supply reliable 
data supporting the general use of an assay to predict patient response to 
chemotherapeutics. 
An in vitro chemoresponse assay is not in general use because of a number 
of obstacles which have contributed to the lack of clinical feasibility of 
the assay. Currently, in vitro drug response assays are performed in 
university hospitals and a few specialized service centers. These 
institutions generally require the transportation of the specimen, 
resulting in a loss of often more than 24 hours before specimen processing 
can begin. Within this 24 hour period, specimen viability declines 
significantly. Additionally, an in vitro assay is not in general use 
because of the lack of truly effective cancer drugs and difficulties in 
trying to model in vivo pharmacokinetics. Other reasons why in vitro 
chemoresponse tests are not in general use include: technical complexity 
of the assays; the inability to grow tumor cells in vitro; long 
turn-around time; large number of tumor cells required to conduct an 
assay; low percentage of specimens suitable for the assay; and the lack of 
quality control for drugs and medium. 
SUMMARY OF THE INVENTION 
The present invention relates to a method for assaying the sensitivity of 
biopsied tumor cells to chemotherapeutic agents, the method comprising 
incubating tumor cells with a sufficient amount of growth medium to form a 
cellular suspension, adding said cellular suspension to a first 
multi-compartment vessel; adding a predetermined amount of at least one 
chemotherapeutic agent in the dry form to a second multi-compartment 
vessel; adding a sufficient amount of said medium to reconstitute said dry 
chemotherapeutic agent to within physiologically reachable dosage ranges; 
adding reconstituted chemotherapeutic agent to certain compartments of 
said first vessel containing tumor cells; incubating said vessel for a 
sufficient period of time for said chemotherapeutic agent to affect said 
tumor cells; adding an indicator of tumor cell viability or growth to said 
first vessel; measuring the amount of said indicator, and comparing the 
amount of said indicator in said compartment to which said 
chemotherapeutic agent was added with the amount of said indicator in the 
compartment that did not receive said chemotherapeutic agents, to 
determine the sensitivity of said tumor cells for said chemotherapeutic 
agent. The first vessels may be coated with a growth matrix to increase 
specimen evaluability. 
The present invention also relates to a kit to assay biopsied tumor cells 
for sensitivity to chemotherapeutic agents comprising: a) a first 
multi-compartment vessel for receiving said biopsied tumor cells; b) a 
second multi-compartment vessel for receiving predetermined amounts of at 
least one chemotherapeutic agent in the dry form; c) a sufficient amount 
of medium to support the growth of said tumor cells in said first 
multi-compartment vessel; d) a sufficient amount of medium to reconstitute 
said dry chemotherapeutic agent to within physiologically reachable dosage 
ranges; e) an indicator of cell proliferation or cell viability; and f) 
means for determining the percent of inhibition of cell growth or 
proliferation as a measure of said biopsied tumor cell sensitivity to said 
chemotherapeutic agents. In this case the first vessel may also be coated 
with a growth matrix to increase specimen evaluability. 
It is an object of this invention to provide a method for assaying the 
sensitivity of biopsied cells to therapeutic agents using a small number 
of tumor cells. 
It is an object of this invention to provide a 96-microwell coated with 
extracellular matrix to increase specimen evaluability and to permit assay 
automation. An extra cellular matrix is used to provide a natural stratum 
enhancing in vivo growth properties and biological responsiveness. 
It is another object of this invention to provide a defined growth medium 
with minimal serum and calcium content to selectively enhance the growth 
and proliferation of epithelial tumor cells. It is still another object of 
this invention to enhance the proliferation of epithelial tumor cells with 
supplemental hormones and growth factors. 
It is still another object of this invention to provide chemotherapeutic 
agents in a microwell drug strip, to facilitate selection of desired 
chemotherapeutics. 
It is still another object of this invention to use tritiated thymidine 
uptake as an indicator of tumor cell viability to reduce the subjectivity 
of cell viability measurements. Tritiated thymidine uptake assay reduces 
specimen testing time from approximately 3 weeks to 5 days. 
It is still another object of the invention to reduce technical complexity 
through the use of prepared drug strips and radionucleotide detection 
which can be automated and which does not require specialized training. 
This reduced technical complexity permits the use of an in vitro 
predictive assay in a clinical laboratory allowing the usage of fresh 
specimens. It is important to note that use of fresh specimens maximizes 
cellular viability.

DETAILED DESCRIPTION OF THE INVENTION 
This kit is used to test the response of individual cancer patient's 
sensitivity and resistance to standard panel of anti-cancer drugs. Tumor 
specimens including those obtained from surgical specimens, malignant 
fluids, bone marrow or blood, are cultured either directly on tissue 
culture plastics, or tissue culture plastics with modified surface (coated 
with one or more extracellular matrices, fibronectin, collagens, or 
others; or surface charge modified by a variety of methods) for recovery. 
The dried drugs are reconstituted with growth medium and transferred to 
the wells containing the cells. The plates are incubated further for drugs 
to express their effect on cells. The growth of cells in drug containing 
wells or control wells (no drugs) is compared. A variety of methods used 
to assess proliferation including radionucleotide incorporation, dye 
reduction, or protein and nuclear stain can be employed. Inhibition of 
growth due to drugs as compared to the 100% controls is used to predict 
the probability of patient's response to drugs. 
This kit is composed of a multi-compartment vessel, coated or not coated 
with a layer of growth matrix. The growth matrix can be either secreted by 
bovine cornea endothelium cells, or reconstituted basement membrane 
proteins or other proteins, matrices which facilitate cell attachment. The 
compartment can also be modified by adding electric charges to facilitate 
cell attachment. 
The usage of the microtiter well, however, facilitates automation. Assay 
technology using chromogenic dyes, fluorogenic dyes can be read with ELISA 
plate reader or fluorogenic reader. In addition, the technique employing 
incorporation of radioactive labelled nucleotide or proteins as an 
indication of cell growth can be simplified by using the microtiter well. 
Incorporated radioactive compound can be harvested with a cell harvester. 
The chemotherapeutic agents can be dried in strips. See FIG. 2. The strips 
fit into a housing with similar configuration as the microtiter well. FIG. 
3. This configuration facilitates drug transfer with multiple channel 
pipettor. Chemotherapeutic agents at different predetermined 
concentrations are dispensed, quickly frozen and lyophilized. Dried drug 
strips are packaged in aluminum foil pouch or other air-and light-tight 
packaging material with desiccant, and sealed. The strip format allows the 
user to select desirable drugs. 
Another feature of the kit is a medium and supplement which support the 
growth of human tumors. Conventionally, drug response assay is done at 
service labs because of the laborious process of drug dilutions, medium 
and matrix preparations and labor intensive method used to obtain results. 
In addition, there is no quality control for the reagents used for the 
test, ie, drugs, medium and medium supplement. This kit simplifies the 
technical complexity and problems and makes it possible to conduct the 
test at most hospital clinical laboratories. 
Strips of microtiter wells are fit into a first multi-compartment vessel 
such as a 96 well microtiter plate. Plates other than 96-well plates may 
be used, ie, 6, 24, or 48 cluster plates or any other formats of multiple 
cluster plates. These plates come in the form of strip plates or the 
conventional culture plates. 
Wells may or may not be coated with a layer of growth matrix. 
Variations in the growth matrix, or tissue culture surface modifications 
include: extracted basement membrane proteins from various sources (ie, 
rat tail collagens, fibronectin, EHS transplantable tumors maintained in 
euthymic nude mice, matrix deposited by normal or tumor cell lines, etc). 
Tissue culture surface may be modified by treating the surface with 
electric corona discharge to facilitate cell attachment. Alternatively, 
matrix may not be used and drugs can be directly deposited on the tissue 
culture plastic surface. 
Anti-cancer drugs are dispensed in replicates, at various predetermined 
concentrations, into the vessels and dried. Drugs may also be dried in 
vessels coated with any variety of matrices. The term "dried" means 
lyophilized or dried at low temperatures. Drugs may be dispensed into 
strips, ELISA plates, or vessels with a variety of configurations or 
tissue culture plastics. The strip format is used for two purposes: 1) to 
offer the user a choice of drugs to be tested; 2) to allow the cells to 
recover after initial processing before challenging cells with drugs. If 
cells do not need to recover, then tissue culture plates with dry drugs 
can be used to save the drug reconstitution and transfer step. In this 
case, cells can be directly plated in tissue culture plates containing the 
dry drugs. If drugs are dried on vessels other than where the cells are 
cultured, then drugs need to be reconstituted and transferred to the plate 
where the cells are cultured. Dried drug strips are packaged in air and 
light tight materials such as aluminum foil pouch with desiccant and 
sealed. 
Specialized medium (RPMI medium with 1% fetal bovine serum), growth factor- 
and hormone-containing supplement (Cyto-Gro.TM. 289 supplement) and growth 
matrix capable of supporting the growth of tumor cells are included with 
the kit. Variations in the growth medium, and supplement growth medium may 
be any enriched buffer medium such as DMEM, F12, McCoy's, CMRL, etc, that 
is suitable for the growth of cells. Growth supplement may be any 
combination of growth factors and hormones included or not included in 
Cyto-Gro.TM. 289 supplement which promote the growth of cells being 
tested. 
Fresh tumor specimens are collected and transported according to standard 
practices. The specimen is processed to obtain a cell suspension using 
mechanical and enzymatic digestion procedures and a differential count 
using trypan blue to assess viability. It should be noted that in the 
presently described procedure there must be at least 3.times.10.sup.5 
tumor cells to conduct the assay. If necessary, tumor cells are enriched 
with the use of Ficoll.TM. and Percoll.TM. density gradient separation 
methods. 
The processed cells are suspended in defined growth medium consisting of 
Roswell Park Memorial Institute (RPMI) 1640 medium, 1% fetal bovine serum 
(FBS), and Cyto-Gro.TM. 289 supplement and aliquoted into an 
extra-cellular matrix coated 96-microwell tissue culture plate. 
Cyto-Gro.TM. 289 supplement was developed by Bartels Diagnostics with the 
goal of providing hormone and growth factor supplements for low serum 
growth medium to optimize epithelial tumor cell growth, thereby 
eliminating the need to inventory each additive separately. Cyto-Gro.TM. 
289 supplement contains insulin, transferring, selenium, B-estradiol, 
hydrocortisone, prostaglandin F2a, and epidermal growth factor. RPMI, 
Cyto-Gro.TM. 289 supplement and a low concentration of serum (1%), combine 
to provide a growth medium with selective advantages for the growth of 
human epithelial tumor cells. 
Maximum proliferation of epithelial cells is also dependent upon the 
presence of a substratum of extracellular matrix. Extracellular matrix is 
composed of different types of collagens, glycosaminoglycans, proteoglycan 
and glycoproteins. The use of extracellular matrix maximizes cell 
attachment and survival and has also been found to optimize the effect of 
hormones and growth factors in defined media. A number of adhesive cell 
matrices have been investigated, both natural and reconstituted. The use 
of naturally produced strata provides a more biologically similar growth 
matrix, assuring that the components are in a native configuration. 
Reconstituted extracellular matrices that have been explored include 
Matrigel.TM., a urea extract from Engelbreth-Holm-Swarm mouse embryonal 
carcinoma, and biomatrix, placental tissue extracted by salt solutions, 
nucleases and detergents. Bovine cornea endothelium cells produce an 
extracellular matrix beneath the cell layer, which is the most commonly 
used extracellular matrix. Research has shown that when cells are grown on 
bovine cornea endothelium cells extracellular matrix monolayers are 
formed, with no aggregates or clumping. Even if cells are seeded as 
aggregates, the cells will spread out, Vladavsky, I., G.M. Lui and D. 
Gospodarowicz, Morphological Appearance, Growth Behavior and Migratory 
Activity of Human Tumor Cell Maintained on Extracellular Matrix Versus 
Plastic, Cell 607-616, March 1980. The formation of cell monolayers, 
promoted by extracellular matrix, aids in the optimization of the 
tritiated thymidine incorporation assay. The use of extracellular matrix 
has been shown to increase the number of specimens successfully cultured 
in vitro, 85-89% versus 59-60%, respectively. With prostate carcinoma, 89% 
of the specimens were evaluable when grown on extracellular matrix, but 0% 
were evaluable when grown on plastic. Renal tumors had an evaluability 
rate of 95% with extracellular matrix and 11% with plastic, Pavelic, K., 
M.A. Bulbul, H.K. Slocum, Z.P. Pavelic, Y.M. Rustum, M.J. Niedbala, and 
R.J. Bernacki, Growth of Human Urological Tumors on Extracellular Matrix 
as a Model for the In Vitro Cultivation of Primary Human Tumor Explants, 
Cancer Research, 46:3653-3662, 1986. Endometrial and ovarian carinoma 
which had not previously been maintained successfully in cell culture were 
100% culturable with extracellular matrix. The increased success rate of 
tumor cell grown on extracellular matrix compared to plastic is a 
manifestation of cells being able to adopt in vivo growth properties by 
becoming physiologically responsive to hormones and growth factors, 
Crickard, Kent, Ulla Crickard, Mahmood Yoonessi, Human Ovarian Carcinoma 
Cells Maintained on Extracellular Matrix Versus Plastic, Cancer Research, 
43:2762-2767, 1983, Pavelic, K., M.A. Bulbul, H.K. Slocum, Z.P. Pavelic, 
Y.M. Rustum, M.J. Niedbala, and R.J. Bernacki, Growth of Human Urological 
Tumors on Extracellular Matrix as a Model for the In Vitro Cultivation of 
Primary Human Tumor Explants, Cancer Research, 46:3653-3662, 1986, 
Vladavsky, I., G.M. Lui and D. Gospodarowicz, Morphological Appearance, 
Growth Behavior and Migratory Activity of Human Tumor Cells Maintained on 
Extracellular Matrix Versus Plastic, Cell 607-616, March 1980, 
Gospodarowicz, Denis, Charles III, Extracellular Matrix and Control of 
Proliferation of Vascular Endothelial Cells, Journal of Clinical 
Investigation, 65:1351-1364, 1980. These characteristics, increased 
evaluability, improved tritiated thymidine uptake due to cell monolayer 
formation, and responsiveness to growth medium components, make 
extracellular matrix especially valuable in a chemosensitivity assay, 
where an in vitro cell response is used as an indication of an in vivo 
tumor response. 
The cells are allowed to recover for 24 hours in 37.degree. C., 5% CO2, 
humidified incubator before they are treated with the drug. Cancer 
chemotherapeutics lyophilized in a strip of 16 wells in a 2.times.8 format 
containing four different amounts of the drug are reconstituted with 
growth medium and added to cells. The four different concentrations are 
based on the plasma achievable level of the drug, along with a higher drug 
concentration to detect extremely resistant cells and two lower 
concentrations to quantity sensitive cells. The drugs that are currently 
available represent commonly prescribed cancer chemotherapeutics: 
Adriamycin.TM., Bleomycin.TM., Cisplatinol.TM. (cis-platin diamine 
dichloride), Etoposide.TM., 5-Fluorouracil.TM., Melphalan.TM., 
Methotrexate.TM., Mitomycin C.TM., and Vinblastine sulfate. 
The cells are incubated with the selected drugs for 3 days allowing the 
drug to express its effect. On the fifth day of post-specimen processing, 
tritiated thymidine at 1 microCurie per well is added. Incorporation of 
tritiated thymidine into nucleotides during DNA synthesis is used as an 
indication of cell growth. After the tumor cells have been exposed to the 
radionucleotide, the cells are harvested and the radionucleotide measured 
with a scintillation counter. The amount of radionucleotide incorporated 
into nucleic acid by the positive control which has not been exposed to a 
drug, is compared to that incorporated by drug treated cells and 
represented as counts per minute (CPM). The growth of resistant tumor 
cells, those not affected by the drug, will be comparable to those in the 
positive control wells and have similar radioactivity. Tumor cells 
sensitive to a drug will have a reduction in growth characterized by a 
reduction in DNA synthesis and demonstrated by a reduction in the uptake 
of tritiated thymidine, represented as CPM, when compared to the positive 
control. See FIG. 1. 
Reagents and Materials 
1. 96-microwell tissue culture plate with extracellular matrix derived from 
bovine cornea endothelium cells packaged in a foil pouch with a molecular 
sieve desiccant 
2. Percoll.TM. isotonic 90% stock solution for gradient cell separation in 
specimen processing in a polyvinylpyrrolidone coated colloidal silica 
suspension, 15 ml (Parmacia Chemicals, Piscataway, N.J.) 
3. Growth media: lyophilized Cyto-Gro.TM. 289 hormone and growth factor 
supplement to be reconstituted with basal media consisting of Roswell Park 
Memorial Institute medium (RPMI), (Life Technologies, Grand Island, 
N.Y.)+1% fetal bovine serum (HyClone Laboratories, Logan, Utah) 
reconstitute to 1 L 
4. L-glutamine (Sigma, St. Louis, Mo.) 2 mM, 5 ml Asparagine (Sigma, St. 
Louis, Mo.) 0.5 mM, 5 ml 
5. Microwell drug strips packaged in a foil pouch with a molecular sieve 
desiccant. See FIG. 2. Each drug strip contains 16 wells in a 2.times.8 
format. The concentrations after reconstitution (.mu.g/ml) are listed 
below and stated on the label of each strip. Available drugs to be tested 
include: 
______________________________________ 
Supplied Concentra- 
Drug Name Vendor tions (.mu.g/ml) 
______________________________________ 
Adriamycin .TM. 
Adria Laboratories 
0.01, 0.1, 1.0, 10.0 
Bleomycin .TM. 
Bristol-Myers 0.01, 0.1, 1.0, 10.0 
Cisplatinol .TM. 
Bristol-Myers 0.01, 0.1, 1.0, 10.0 
Etoposide .TM. 
Bristol-Myers 0.05, 0.5, 5.0, 50.0 
5-Fluorouracil .TM. 
Smith and Nephew 
0.1, 1.0, 10.0, 100.0 
Methotrexate .TM. 
Quad Pharmaceuticals 
0.01, 0.1, 1.0, 10.0 
Mitomycin-C .TM. 
Bristol-Myers 0.01, 0.1, 1.0, 10.0 
Vinblastine .TM. 
Quad Pharmaceuticals 
0.01, 0.1, 1.0, 10.0 
______________________________________ 
6. Ficoll.TM., 100 ml (LSM manufactured by Organon Teknika Corporation, 
Durham, N.C.) 
Storage of Materials and Reagents 
96-microwell extracellular matrix tissue culture plates, growth medium, and 
Percoll.TM. were stored at 2.degree.-8.degree. C. Drug strips were stored 
unopened at 2.degree.-8.degree. C. Ficoll.TM. and lysing buffer were 
stored at room temperature. Glutamine, pyruvate and asparagine were stored 
below -20.degree. C. Reagents were brought to room temperature before use. 
Specimen Collection and Transport 
Surgical Specimens 
Fluid Specimen Preparation 
Ascites fluid was tapped into a vessel containing preservative-free sodium 
heparin (Invenex Laboratories Division of LyphoMed, Melrose Park, Ill. 
60160) 10 units/ml ascites final volume. It should be noted that the 
presence of preservatives may inhibit the growth of tumor cells, and 
therefore should not be used to collect samples for use in this assay. 
Fluid specimens may remain unprocessed for 24-48 hours. For best results, 
however, begin the assay immediately. 
Solid Specimen Preparation 
Tumor tissue was handled aseptically at all times. For better results, the 
viable area of the tumor tissue should be isolated by trimming off fat and 
normal tissue, avoiding necrotic sections. 10 ml of transport medium (10% 
FBS in minimal essential medium (MEM) 500 ml) pipeted to a 50 ml conical 
tube was added to the trimmed tumor tissue. For better results, surgical 
specimens were minced to 1 mm within 30 minutes of removal. The assay 
should be begun immediately. If necessary, minced specimens may be held up 
to 16 hours after surgery, but no longer than 24 hours. 
Specimen Preparation and Processing 
Solid Specimen Processing 
All specimen preparation steps were conducted in a laminar flow hood using 
aseptic technique. The specimen was removed from the transport medium 
using sterile forceps and placed in a 100 mm petri dish containing 5 ml 
tissue culture medium; RPMI+10% FBS. The specimen was minced, using a 
sterile scalpel, into pieces less than 1 mm in size. The tissue pieces 
were rinsed with 10 ml tissue culture medium and the medium was 
transferred, avoiding the larger tissue pieces, to a polystyrene tube. The 
tissue was washed repeatedly with 1 ml fresh tissue culture medium and 
then the rinses were added to a polystyrene tube. The number of tumor 
cells were counted. If an adequate cell number is obtained (see "Plating 
Density" section below), further processing was not necessary. If not, the 
sample should be processed further using the following steps. Transfer the 
minced specimen to a Cellector.TM. tissue sieve and gently force the 
specimen through the sieve by pressing downward with a glass pestle. 
Combine the tissue rinse to the tissue and medium collected from the 
tissue sieve and wash twice with tissue culture medium. If the tissue is 
too fibrous or collagenous and the cells cannot be dispersed by mechanical 
means such as the Cellector.TM. tissue sieve, digestive enzymes should be 
used to free single cells. A typical enzyme digestion includes use of a 
medium containing 0.08% collagenase and 0.002% DNase, incubating for 1-18 
hours at 37.degree. C. with gentle shaking. Cell viability should be 
monitored at 30 minute intervals for the first 2 hours. Viability should 
increase as the outer dead cells and connective tissue are digested. Allow 
the large pieces of tissue to settle and transfer the medium with 
suspended cells to a polystyrene conical tube. Wash the cells twice with 
tissue culture medium to remove enzymes, resuspending the cells in growth 
medium. 
Tumor cell viability was assessed using a method such as a differential 
count using trypan blue and a hemacytometer. The total viable tumor cell 
count in the present assay must be at least 1-5.times.10.sup.5 cells to 
test one drug plus controls. More cells were necessary for each additional 
drug tested. 
After mechanical and enzymatic cell dispersion, the specimen may still 
required treatment to separate the tumor cells from the normal available, 
such as magnetic beads, Ficoll.TM. and/or Percoll.TM. gradients. The 
Ficoll.TM. and Percoll.TM. gradient cell separation methods are described 
here. If the cell suspension does not require further treatment, prepare 
the cell suspension to contain 1-5.times.10.sup.5 cells/ml in growth 
medium. Eleven milliliters of the cell suspension were required in the 
present assay for each 96-well plate inoculated. 
Fluid Specimen Preparation 
All specimen preparation was conducted in a laminar flow hood using aseptic 
technique. The ascites fluid was mixed to achieve an even cell suspension 
by swirling. Only the fluid was transferred to centrifuge tubes and 
centrifuged at 400.times.g for 7 minutes. The supernatant was removed. 
The cell pellet was resuspended and washed in growth medium, and 
centrifuged at 400.times.g for 7 minutes. All pellets were combined and 
resuspended in growth medium, Cyto-Gro.TM. 289 supplement+1% FBS+RPMI. 
Tumor cell viability was assessed using a method such as a differential 
count using trypan blue and a hemacytometer. The total viable tumor cell 
count must be at least 3.times.10.sup.5 cells, for the present assay, to 
test one drug plus controls. More cells were necessary for each additional 
drug tested. Using the results of the differential count, the cell 
suspension was prepared. Eleven milliliters of the cell suspension were 
required for each 96-well plate inoculated. 
Tumor Cell Enrichment 
Ficoll.TM. Cell Separation- Note that this step is necessary if specimen 
contains more than 20% red blood cells. 
Ficoll.TM. specimen processing was conducted in a laminar flow hood using 
sterile technique. 4 ml aliquots of Ficoll.TM. solution was dispensed 
(specific gravity 1.077) into conical polystyrene tubes. The specimen was 
suspended in tissue culture medium containing 10% fetal bovine serum (FBS) 
to attain a viable tumor cell. 10 ml of the specimen suspension was 
layered over the 4 ml aloquot of Ficoll.TM.. Note that these two solutions 
should not be mixed. The suspension was centrifuged at 1000.times.g for 15 
minutes. The cell layer at the interface of the two solutions was removed 
and transferred to a clean conical polystyrene tube. The cell layer was 
centrifuged at 400.times.g for 7 minutes. The supernatant was discarded 
and the cell pellets were resuspended, washing the cells these times with 
tissue culture medium. The cell pellet was resuspended in the tissue 
culture medium and the viable tumor cells were counted using a 
differential count method such as trypan blue and a hemacytometer. Using 
the results of the differential count, a cell suspension was prepared to 
contain 3.times.10.sup. 5 cells/ml in growth medium. Eleven milliliters of 
the cell suspension was needed for each 96-well plate inoculated. 
Percoll.TM. Gradient Cell Separation:- This step necessary if the specimen 
has a high percentage lymphocytes (.gtoreq.30%) and/or a high amount of 
cellular debris. 
Percoll.TM. separation was conducted in a laminar flow hood using sterile 
technique. The Percoll.TM. solution was diluted at 90% to 10% and 20% 
using tissue culture medium. 10% and 20% Percoll.TM. solutions were 
prepared just prior to use. The number of gradients required for the 
entire specimen was calculated as follows. Each gradient can accommodate 
2-3 ml containing up to 2.times.10.sup.7 total cells. Prepare a 10%-20% 
Percoll gradient by aliquoting 4 ml of 10% Percoll.TM. in a polystyrene 
conical centrifuge tube. 4 ml of the 20% Percoll.TM. was layered under the 
10% Percoll.TM., being careful not to disturb the upper Percoll.TM. layer. 
The cell suspension obtained from the Ficoll.TM. separation was layered 
onto the Percoll.TM. gradient. The cellular suspension was centrifuged at 
50-60 xg for 10 minutes at room temperature. 
The majority of the tumor cells were observed to be pelleted in the bottom 
of the centrifuge tube. The lowest interface in the tube, however, may 
contain some tumor cells, that can be collected and inspected if desired. 
The higher interface contained mainly normal cells, such as leukocytes and 
may be discarded. The selected cell fractions were washed three times and 
resuspended with tissue culture medium. The tumor cells were counted using 
a differential cell count method such as trypan blue and a hemacytometer. 
A minimum viable tumor cell count of 3.times.10.sup.5 cells was required 
to test one drug. More cells were required for additional drug testing. 
Using the results of the differential count, the cell suspension was 
prepared to contain 3.times.10.sup.5 cells/ml of growth medium. Eleven 
milliliters of cell suspension was found to be required for each 96-well 
plate inoculated. 
Positive and Background Assay Control 
For each specimen, two columns of 16 wells were set aside for background 
and positive controls. The first column of 8 wells (1) were background 
control wells which remained cell- and drug-free serving as a baseline 
radioactivity control. As a result, the background control wells indicated 
how well the wash step removes tridiated thymidine from an extracellular 
matrix well. The positive control wells were the second column of 8 wells 
(2) and were inoculated with tumor cells, but remained drug-free. The 
positive control wells provided tritiated thymidine uptake data from a 
tumor cell population not treated with cancer chemotherapeutics. 
Plating Density 
Plating density per well were 1-2.times.10.sup.4 viable tumor cells per 
well for large cells such as ovarian and mesothelioma tumor cells, and 
3-5.times.10.sup.4 viable tumor cells per well for small tumor cells such 
as small cell lung. Intermediate size tumor cells such as colon, prostate, 
bladder, breast and lung should be plated between 2-3.times.10.sup.4 
viable tumor cells per well. 
Plating Density Example: 
If 5 drugs are tested, the total tumor cells requirements are as follows: 
Controls: 8 wells.times.1-5.times.10.sup.4 cells/well/100 
.mu.l=0.8-4.times.10.sup.5 cells 
5 drug strips: 80 wells.times.1-5.times.10.sup.4 cells/well/100 
.mu.l=8-40.times.10.sup.5 cells 
Total cells required=8.8-44.times.10.sup.5 cells 
Cell Inoculation 
Cell inoculation was conducted in a laminar flow hood using sterile 
technique. The number of extracellular matrix microtiter plates required 
was calculated as follows. If one drug is tested, 24 wells must be 
inoculated, if two drugs are tested, 40 wells must be inoculated, etc. 
Positive and background control wells must be run with each specimen. 
Positive the background control wells in column 1, and the positive 
control wells in column 2 (FIG. 1). 
The cells were evenly dispersed by inverting the tube with growth medium 
and cells 5-6 times. Inoculations were facilitated with the use of a 
multi-channel pipettor and V-shaped reservoir (Costar.TM.-Cambridge, 
Mass.). It is important that the dispensing procedure be completed in less 
than one minute to ensure an even distribution of cells. The positive 
control wells (column 2) were inoculated with cells. The background 
control wells were not inoculated with cells (column 1). 100 .mu.l of 
growth medium containing 1-5.times.10.sup.5 cells/ml of the tumor cell 
suspension was pipetted into the appropriate wells (1-5.times.10.sup.4 
cells per well total). The background control wells were not inoculated 
with cells. Note that pipet tips were changed for every cell transfer to 
decrease the risk of contamination. A multichannel pipettor facilitates 
ease of inoculation. Extracellular matrix plates were covered and 
incubated at 37.degree. C. with 5% carbon dioxide for 24 hours in a 
humidified environment. 
Drug Addition 
The foil packaged drug strips were allowed to reach room temperature before 
opening. The package was opened in a laminar flow hood. The micro drug 
strips were removed from the foil pouch and placed in the plate frame with 
the labelled end at the bottom of the frame and snapped firmly into place. 
FIG. 3. The strip label was coded according to the particular drug. The 
control strip, labelled CS, was placed in columns 1 and 2 and the drug 
strips to be assayed in subsequent columns. Four concentrations of the 
drug are contained in each 2.times.8 drug strip. The bottom 4 wells 
contained the lowest and the top 4 wells the highest drug concentration 
(see diagram below). 
After placing all drug and control strips in the plate frame, (FIG. 3) 125 
.mu.l of growth medium was added to each well. When reconstituting 
lyophilized drug, start with the lowest drug concentration wells and 
progress to the highest drug concentration. Pipet tips were changed before 
each medium addition. When assaying more than five drugs, a second plate 
frame is required. Additional control strips are not required for the 
second plate. 
A homogeneous suspension was ensured by allowing the drugs to solubilize 
for five minutes and mixing by repeated aspirations. 100 .mu.l of the 
reconstituted drugs was pipetted to the appropriate wells containing 
cells. Note: do not inoculate control wells. 
If testing more than one drug, pipet tips should be changed between drug 
strips. The vessels were covered, plated, incubated and humidified at 
37.degree. C., 5% carbon dioxide for three days, except when testing 
5-Fluorouracil.TM., a longer incubation time (&gt;96 hours), and thus, a 
separate control must be used. 
Tritiated Thymidine Assay 
Prior to adding tritiated thymidine, the compartments of the vessel were 
observed for wells with consistent cell layer growth, microbial 
contamination and for wells which show toxic cells. 
The amount of tritiated thymidine that was needed to run the assay was 
calculated: 25 .mu.l of tritiated thymidine was added to each well to 
achieve a final concentration per well of 1.0 microCurie. To achieve this, 
prepare a 1:25 dilution of stock tritiated thymidine (6.7 ci/mMol; 1.0 
milliCurie/ml) in growth medium, resulting in a working strength 
concentration of 40 microCurie/ml. The addition of 25 .mu.l to a well will 
result in a concentration of 1microCurie/well. If an entire 96-well plate 
is assayed, 96.times.25 .mu.l or at least 2.4 ml of the 1:25 dilution will 
be needed (i.e., 104 .mu.l stock tritiated thymidine+2.5 ml growth media). 
Preparation and addition of the tritiated thymidine: The appropriate amount 
of growth medium and tritiated thymidine was added to a tube. Pipet 25 
.mu.l of the 40 microCurie/ml dilution was pipetted to each compartment, 
changing tips after each transfer. The plate was covered and incubated in 
a humidified environment at 37.degree. C. 5% carbon dioxide for 10 to 16 
hours. 
Cell Harvesting 
The harvester was prepared by washing the lines with approximately 40 ml 
70% ethanol. The filter paper was placed in the harvester and the punch 
lowered. The filter paper was prewet by running approximately 25 ml water 
through the harvesting head. The plate to be harvested from the incubator 
was removed, and the harvesting head was placed into the first two rows, 
aspirating the contents of the wells. Water was dispensed through the 
harvesting head and washed until approximately four well volumes have 
passed (1 ml). 100 .mu.l of lysing buffer was added to these 24 wells, 
leaving the buffer on for two minutes. 
The wells were thoroughly washed with water through the harvesting head 
with 6 to 8 well volumes (1.25 to 2 ml). 
The harvesting head lines were cleaned with approximately 40 ml 70% 
ethanol. The harvester vacuum was switched to reverse and the filter 
striphead opened. 
The 24 individual filters were placed into scintillation vials. 
Steps 1 through 9 were repeated until all wells were harvested. 
Approximately 2 ml scintillation fluid was added to the vials, capped and 
read in the scintillation counter. 
Definition of evaluable samples 
As assay is considered evaluable only if the following criteria are 
satisfied: the average CPM of the untreated control is .times.1000 CPM; 
the average CPM is four times above the background CPM; the background CPM 
is &lt;200 CPM; the coefficient of variation for the 8 control wells 
((control standard deviation/control average).times.100%) is &lt;50%. 
Data Interpretation 
The background control value was the mean of the radioactivity counts from 
the 8 background control wells containing medium but no cells. This value 
served as a baseline radioactivity control to ensure that adequate 
harvester washes were achieved. 
The positive control value was the mean of the radioactivity counts from 
the 8 positive control wells. Cells were added to the wells, but not 
chemotherapeutics and therefore the tritiated thymidine uptake by these 
cells represent maximum incorporation. The counts per minute (CPM) from 
the positive controls were considered 100%. 
Test results were represented here as % control, calculated by dividing the 
CPM from drug treated wells with CPM from control wells, as below: 
##EQU1## 
A range of plasma and 1/10 plasma achievable drug concentrations are 
presented, represented by the hatched portion of the graph. SEE FIG. 4-8. 
For data interpretation, the % control value corresponding to the mean 
value of 1/10 plasma achievable drug concentration is used. Drugs which 
resulted in less than or equal to 20% of control growth, when used at 1/10 
plasma achievable concentration, is considered sensitive. Drugs which 
inhibited less than 20% growth, having a CPM above 20% of the control 
value are considered resistant. 
20% control (80% inhibition) is used as the positive/negative cut off value 
as it has been found to more closely correlate with clinical responses in 
similar in vitro drug response assay. David H. Kern, et al., Development 
of a Miniaturized, Improved Nucleic Acid Procedure Incorporating Assay for 
Chemosensitivity Testing of Human Solid Tumor, 45 Can. Res., 5436 (1985), 
Tanigawa, Nobuhiko, David H. Kern, Yorinori Hikase, and Donald L. Morton, 
Rapid Assay for Evaluating the Chemosensitivity of Human Tumors in Soft 
Agar Culture, Cancer Research, 42:2159-2164, 1982. ChemoResponse Assay 
results obtained from a surgical breast specimen have been plotted in 
FIGS. 4-8. The results indicate that the specimen is sensitive to 
Adriamycin, Bleomycin and 5-Fluorouracil (FIGS. 4-6) and resistant to 
Cisplatinol and Melphalan (FIGS. 7 and 8). 
Although the invention has been shown in connection with certain specific 
embodiments, it will be readily apparent to those skilled in the art that 
various changes in form and arrangement of steps can be made to suit 
requirements without departing from the spirit and scope of the invention.