Method for establishing a tumor-cell line by preparing single-cell suspension of tumor cells from tumor biopsies

The present invention provides a novel method for the establishment of tumorigenic cell lines from biopsies of human or animal tumors. The method includes preparing a single-cell suspension of a tumor cells by injecting cell culture medium into an isolated tumor or a portion thereof to flush out the tumor cells. The tumor or portion thereof may be in a cell culture maintenance medium. The step of injecting cell culture medium into the isolated tumor or portion thereof involves piercing the tumor or portion thereof with a needle to inject the cell culture medium and flush out the tumor cells. The steps of piercing the tumor or portion thereof with a needle and injecting the cell culture medium to flush out the tumor cells preferably are repeated until the single-cell suspension has a particular cell density. The method also includes establishing a primary tumor cell culture by growing the cells from the single-cell suspension in a cell culture medium.

FIELD OF INVENTION 
The present invention relates to a technique to culture in vitro primary 
tumor samples. This technique is different from the standard protocol in 
that it obviates the laborious procedure of chopping and mincing the tumor 
tissue and treating it with enzymes. In the present invention, the cells 
are flushed out of the tumor tissue and allowed to grow in vitro, as a 
single-cell suspension. The tumor cell lines resulting from the method of 
the present invention are superior to the tumor cell lines resulting from 
the standard protocol in that they are more tumorigenic. Using the method 
of the invention, new potential anticancer drugs for the treatment of 
cancers such as adenocarcinomas (more specifically, adenocarcinoma of the 
colon, lung, breast, prostate, ovary, pancreas, duodenum, intestines, 
kidney and liver) or for treatment of cancers characterized by loosely 
bound tumor cells can be tested more efficiently. 
The in vitro and in vivo models described herein are of great value in 
identifying novel anticancer agents and developing new treatment 
strategies for cancer. Establishment of primary tumor cell cultures 
facilitates identification and characterization of the key growth factors 
responsible for uncontrolled growth and, thereby, facilitates designing 
cancer cell targeted therapy. The primary tumor cell cultures are 
established by optimizing the procedure for isolation of cells and culture 
conditions to achieve continuous growth of the primary tumor cells that 
represent most of the sub-population of cells present in the tumor in 
situ. Transfer of primary tumor cells to laboratory animals further aids 
in testing new anticancer agents. 
BACKGROUND 
Reports in the scientific literature disclose that primary tumor cultures 
have been established by chopping and mincing the tumor tissue and 
treating it with various enzymes to finally grow it as an explant (Peters, 
et al., "Preparation of Immunotherapeutic Autologous Tumor Cell Vaccines 
from Solid Tumors," Cancer Research 39 (1979):1353-60). This method of 
enzymatic digestion is not very efficient and is prone to contamination 
(Vose, "Separation of Tumor and Host Cell Populations from Human 
Neoplasm," in Cancer Cell Organelles, eds. E. Reid, G. M. W. Cook, and D. 
Y. Morre (Chichester, United Kingdom: Horwood, Ltd., Publishers, 
(1981):45-56). 
Colon cancer tumor induction in nude mice has been previously reported with 
a success rate of 60-80% (Giovanella, et al., "Correlation Between 
Response to Chemotherapy of Human Tumors in Patients and in Nude Mice," 
Cancer 52 (1983):1146-52). The method described in the Giovanella article 
involved the xenografting of primary human tumor tissue in nude mice 
followed by transfer of the cells from the tumor to a culture after the 
tumor tissue had been passaged 2-3 times in the mice. Various routes have 
been used for induction of tumors in nude mice with varying success rates 
(Giavazzi, et al., "Metastatic Behaviour of Tumor Cells Isolated from 
Primary and Metastatic Human Colorectal Carcinomas Implanted into 
Different Sites in Nude Mice," Cancer Research 46 (1986):1928-33). The 
orthotopic transplantation of histologically intact tissue of human 
pancreatic cancer and human metastatic colon cancer in nude mice has also 
been reported (Fu, et al., "A Metastatic Nude-Mouse Model of Human 
Pancreatic Cancer Constructed Orthotopically with Histologically Intact 
Patient Specimens," Proc. Natl. Acad. Sci. U.S.A. 89 (1992):5645-49; Fu, 
et al., "Models of Human Metastatic Colon Cancer in Nude Mice 
Orthotopically Constructed by Using Histologically Intact Patient 
Specimens," Proc. Natl. Acad. Sci. U.S.A. 88 (1991):9345-49). The 
following table A lists in vitro and in vivo models for different cancer 
types. 
TABLE A 
______________________________________ 
Tumor Type 
Model Reference 
______________________________________ 
Pancreatic cancer 
In vivo Fu, et al., "A Metastatic Nude-Mouse 
Model of Human Pancreatic Cancer 
Constructed Orthotopically with 
Histologically Intact Patient 
Specimens," Proc. Natl. Acad. Sci. 
USA 89 (1992): 5645-49. 
Colorectal cancer 
In vivo and 
Giavazzi, et al., "Metastatic Behaviour 
in vivo of Tumor Cells Isolated from Primary 
and Metastatic Human Colorectal 
Carcinomas Implanted into Different 
Sites in Nude Mice," Cancer Research 
46 (1986): 1928-33. 
Colon cancer 
In vivo Fu, et al., "Models of Human Metastatic 
Colon Cancer in Nude Mice Ortho- 
topically Constructed by Using 
Histologically Intact Patient 
Specimens," Proc. Natl. Acad. Sci. 
USA 88 (1991): 9345-49. 
Breast cancer 
In vitro and 
Rae-Venter, et al., "Growth of Human 
in vivo Breast Carcinomas in Nude Mice and 
Subsequent Establishment in Tissue 
Culture," Cancer Research 40 (1980): 
95-100. 
Melanoma, In vivo Giovanella, et al., "Correlation Between 
colorectal cancer, Response to Chemotherapy of Human 
breast carcinoma Tumors in Patients and in Nude Mice," 
Cancer 52 (1983): 1146-52. 
______________________________________ 
SUMMARY 
The invention includes a method of preparing a single-cell suspension of 
tumor cells by injection of cell culture medium into an isolated tumor or 
a portion thereof to flush out the tumor cells. The tumor or portion 
thereof may be maintained in a cell culture medium. The step of injecting 
cell culture medium into the isolated tumor or portion thereof involves 
piercing the tumor or portion thereof with a needle to inject the cell 
culture medium and flush out the tumor cells. The term "flush out" 
incorporates the event of the fluid coming out of the tumor or portion 
thereof after injection into the tumor or portion thereof of cell culture 
medium, wherein the fluid coming out of the tumor or portion thereof 
contains the cells released from the tumor. The steps of piercing the 
tumor or portion thereof with a needle and injecting the cell culture 
medium to flush out the tumor cells preferably are repeated until a 
single-cell suspension is obtained having a particular cell density. 
The invention also includes a method of establishing a primary tumor cell 
culture, the method including the steps of: (a) preparing a single-cell 
suspension according to the method described in the previous paragraph; 
and (b) growing the cells from the single-cell suspension in a cell 
culture medium. The cells grown in step (b) may be passaged.

DETAILED DESCRIPTION 
According to this invention, a tumor sample is obtained. The tumor sample 
is preferably obtained directly from a patient or an animal, although the 
tumor sample obtained from a patient or an animal can be stored for up to 
approximately six months before use. It is preferred that the sample be 
stored for less than six months before use. Before use, the fatty tissue 
and necrotic tissue from the tumor sample are removed. The remaining 
tissue is incubated in a cell culture medium, and then the tissue is cut 
into small pieces (e.g., ranging in size from about 1.times.1.times.1 mm 
to about 10.times.10.times.10 mm and preferably about 5.times.5.times.5 mm 
in size). Cell culture medium that can be used comprises RPMI, fetal calf 
serum, gentamycin, and streptomycin. Nystatin may also be added to the 
cell culture medium. Other culture medium such as DMEM (Dulbeccos Modified 
Essential Medium), F12 Medium, Hanks Balanced Salt Solution, HAM Medium, 
buffered saline or any other suitable cell culture medium may be used. The 
tumor cells are then released and dispersed from the tumor tissue by 
piercing it with a needle and injecting cell culture medium into the 
tumor. It is preferred that the tumor is injected by filing a 5 ml or 10 
ml syringe with approximately 5 ml of cell culture medium and piercing the 
tumor tissue about halfway through using a needle preferably a 21 gauge 
needle 1/2 inch or 1 inch in length and injecting medium into the tissue 
slowly. The fluid coming out of the tumor contains the cells released from 
the tumor. This process results in the dislodgment of cells from the tumor 
tissue which come out with the cell culture medium. The tumor cells are 
slowly flushed out of the tissue, and a single cell suspension is 
obtained. This procedure may be repeated several times until a cell 
density of at least approximately 10.sup.3 cells/ml is obtained. The 
viability of the tumor cells in the single cell suspension may be checked 
by trypan blue. 
To establish a primary tumor cell culture, cells in the single cell 
suspension are plated in culture dishes containing cell culture medium. 
The cultures are maintained in an incubator, wherein the concentration of 
CO.sub.2 and O.sub.2 may be kept constant. The cell debris present in the 
cultures should be gently removed as necessary, and fresh cell culture 
medium should be added regularly. Small colonies will start to form; and 
these colonies subsequently will increase in size to merge with each 
other. When confluence of the colonies is attained, the cells may be 
subcultured. 
The cells are subcultured by incubating them with trypsin (e.g., 0.25% 
trypsin in the cell medium) in the incubator for a period of time ranging 
from approximately 5 minutes to approximately 15 minutes (preferably 
approximately 10 minutes). The effect of trypsin is arrested by adding 
fresh fetal calf serum (e.g., 10%). Then the cells are scraped off the 
culture surface and transferred to a plate with multiple wells. 
Approximately 1000 to 100,000 cells are added to each well. After a period 
of time ranging from approximately 5 days to approximately 10 days 
(preferably 7 days), the cultured cells are transferred from the plate to 
a medium-sized flask and then to a large-sized flask. The expanded 
culture, which is maintained in the large-size flask, constitutes the 
"first passage." Approximately every 10 days, the cells may be passaged 
again. The true identity of the passaged cells may be routinely monitored 
by indirect immunofluorescence using as a probe a tumor specific 
monoclonal antibody. 
The ability of the primary tumor cell culture to form colonies in soft agar 
is a characteristic feature of transformed cells; and, therefore, the 
presence of tumor cells may be checked by growing the cells in soft agar 
in the conventional way. The following protocol, for example, may be used. 
The soft agar is prepared by conventional methods. For instance, an 
approximately 1% solution of tissue culture grade agarose may be prepared 
by melting it at approximately 90.degree. C. for approximately two hours 
and subsequently sterilizing it by gamma irradiation. Healthy primary 
tumor cells are counted and suspended in double strength RPMI 1640 medium 
such that 2 ml contains approximately 50 to 200 cells, preferably 100 
cells. The concentration of fetal calf serum (FCS) in the medium is kept 
at approximately 20%. 2 ml of this cell suspension was plated in each well 
of a multiple-well culture plate and then an approximately equal volume of 
1% agar solution was added to each well. The final concentration of 
solidified agar is approximately 0.5%, the final concentration of fetal 
calf serum is approximately 10%, and the final concentration of RPMI 1640 
is approximately normal strength. The cultures then are incubated at 
approximately 37.degree. C. in a 5% CO.sub.2 incubator. The cultures are 
fed with regular cell culture medium at intervals of approximately three 
to four days to prevent the agar from drying out. If the primary tumor 
cells have the ability to form colonies in soft agar, small colonies will 
become visible and slowly will increase in size; and if left, eventually 
the colonies will start merging with each other. 
Although a few exemplary embodiments of this invention are described in 
detail in the following sections, those skilled in the art will readily 
appreciate that many modifications are possible in the exemplary 
embodiments without materially departing from the novel teachings and 
advantages of this invention. Accordingly, all such modifications are 
intended to be included within the scope of this invention. 
In Vitro Studies 
Tumor tissue biopsies were collected from 12 patients who were diagnosed 
with colon cancer on the basis of clinical history, fine needle aspiration 
cytology (FNAC), and histopathology. Out of the twelve patients, seven were 
males and five females. Their ages were in the range of 47 to 74 years. 
Five out of twelve patients had cancer of the ascending colon; five had 
cancer of the descending colon; and two had cancer of the transverse 
colon. 
For each patient, a tumor biopsy was taken from the periphery of the tumor 
that contained roughly the highest proportion of tumor cells and that was 
not in direct contact with fecal matter. The biopsy was collected in a 
cell culture medium comprising RPMI 1640 (Biological Industries, Israel), 
5% fetal calf serum (Biological Industries, Israel), gentamycin (100 
ug/ml, Fulford), and streptomycin (650 .mu.g/ml, Sarabhai Chemicals, 
India). The biopsy tissue was transported from the operation theater to 
the culture room at approximately 4.degree. C. Cultures were initiated 
within approximately 4 to 12 hours of biopsy collection. 
The tumor biopsy was transferred to a cell culture medium comprising RPMI 
1640 (Biological Industries, Israel), 5% fetal calf serum (Biological 
Industries, Israel), gentamycin (100 ug/ml, Fulford), streptomycin (650 
.mu.g/ml, Sarabhai Chemicals, India) and nystatin (0.1 .mu.g/ml) in an 
aseptic laminar hood. The fatty tissue and the necrotic tissue were 
removed. The remaining tissue was then incubated in the cell culture 
medium for at least ten minutes and for not more than 40 minutes. It is 
preferred that the tissue is incubated for approximately 30 minutes. After 
the tissue is incubated it is (hen transferred to fresh cell culture medium 
comprising RPMI 1640 (Biological Industries, Israel), 5% fetal calf serum 
(Biological Industries, Israel), gentamycin (100 .mu.g/ml, Fulford), 
streptomycin (650 .mu.g/ml, Sarabhai Chemicals, India) and nystatin (0.1 
.mu.g/ml). After four to five such serial transfers, the tissue was used 
for the preparation of a single cell suspension. The tissue was cut into 
small pieces of about 5.times.5.times.5 mm in size. Additional serial 
transfers can take place before the tissue is cut into small pieces. The 
cells were then released and dispersed from the tumor tissue by piercing 
the tumor tissue with a 21-gauge needle preferably 1/2 inch or 1 inch in 
length and injecting cell culture medium comprising RPMI 1640, 10% fetal 
calf serum (FCS), gentamycin (50 .mu.g/ml,) and streptomycin (325 
.mu.g/ml) into the tissue. The cells were slowly flushed out of the tissue 
and a single cell suspension was obtained. This procedure was repeated 
several times until a cell density of at least approximately 10.sup.3 
cells/ml was obtained. The fluid coming out of the tumor contains the 
cells released from the tumor. (The remaining tissue was cut into small 
pieces and stored at -70.degree. C. in neat FCS containing 10% dimethyl 
sulfoxide. Tumor tissue stored in this way can be used for establishment 
of in vitro cultures even after 6 months although it is preferred that the 
tissue be used before 6 months.) Before the tumor cells were plated, the 
cell viability of the single cell suspension was checked by trypan blue 
and was found to be between approximately 80-90%. 
Approximately 10.sup.3 cells/ml were plated on day 0 in 55 mm petri dishes 
containing the cell culture medium that was injected into the tumor 
tissue. The cultures were maintained at approximately 37.degree. C. in a 
CO.sub.2 incubator, with the concentration of CO.sub.2 kept constant at 
approximately 5% and the concentration of O.sub.20 kept constant at 
approximately 95%. The cell debris present in the cultures was gently 
removed by conventional methods, and fresh cell culture medium was added 
to the cultures every third day. Cell growth was observed within 
approximately 24-48 hours of plating, and the cells started adhering to 
the culture surface. It was observed that approximately 95% of the cells 
had a round morphology, a translucent cytoplasm, and a large nucleus 
containing single nucleolus. These observations were consistent with the 
histology of primary tumor cells of human colon adenocarcinoma. By 
approximately days 5-7, small colonies started forming; and these colonies 
subsequently increased in size to merge with each other by approximately 
day 9. Confluence was attained by approximately day 10, when the cells 
were subcultured. 
On day 10 when the cells were subcultured, the cells were incubated at 
approximately 37.degree. C. with 0.25% trypsin for 10 minutes in a 
CO.sub.2 incubator. This incubation step can take place on any of days 5, 
6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The effect of trypsin was arrested 
by adding fresh 10% FCS. The cells were scraped off the culture surface, 
counted, and expanded by transferring them to 6-well plates. After one 
week, the cultured cells were transferred from 6-well plates to 25 
mm.sup.3 flasks and then the cells from the multiple 25 mm.sup.3 flasks 
were combined into a 75 mm.sup.3 flask. The expanded culture, which was 
maintained for 4 to 10 days in the 75 mm.sup.3 flask, was designated 
"first passage". These were then passaged every 10 days and the medium was 
changed between day 3 and 6. The cell culture medium that was used 
comprised RPMI 1640, 5% fetal calf serum, gentamycin (100 ug/ml), 
streptomycin (650 .mu.g/ml) and nystatin (0.1 .mu.g/ml). Six out of the 
twelve primary tumor tissues that were established as cultures were 
passaged in vitro 38 times, four of them were passaged 12 times, and the 
remaining two of them were passaged six times each. In all passages the 
cell culture medium is changed every 3 to 6 days. 
The identity of the passaged cells was routinely monitored by indirect 
immunofluorescence using as a probe an adenocarcinoma specific monoclonal 
antibody that recognizes a 90 kD glycoprotein on the membrane. 
Approximately 90% of the tumor cells stained with the adenocarcinoma 
specific marker as revealed by bright fluorescence on the periphery of the 
tumor cells, when viewed under the UV light. Thus, the reactivity of the 
cultured cells to the monoclonal antibody when checked at different 
passages of the culture was found to be adenocarcinoma positive. 
The identity of the cells as tumor cells was also checked by growing the 
cells which had been passaged at least 5 times in vitro in soft agar. An 
approximately 1% solution of tissue culture grade agarose was prepared by 
melting it at approximately 90.degree. C. for approximately two hours and 
subsequently sterilizing it by gamma irradiation. Healthy primary tumor 
cells were counted and suspended in double strength RPMI L1640 medium such 
that 2 ml contained approximately 100 cells. The concentration of fetal 
calf serum in the medium was kept at approximately 20%. 2 ml of this cell 
suspension was added to each well in a six-well culture plate. The cell 
suspension was plated and an approximately equal volume of 1% agar 
solution was added to the plated cell suspension. The final concentration 
of solidified agar was approximately 0.5%, fetal calf serum was 
approximately 10%, and RPMI 1640 was approximately normal strength. The 
cultures then were incubated at approximately 37.degree. C. in a 5% 
CO.sub.2 incubator. The cultures were fed with regular cell culture medium 
at intervals of approximately three to four days. Small colonies of cells 
became visible after approximately 5-7 days. Approximately 0.5 ml of the 
cell culture medium was added on top of the agar to prevent the drying up 
of the agar. When the colonies became visible, the cells had a round, 
translucent morphology. The colonies slowly increased in size, and by day 
30 they started merging with each other. The primary tumor cells of human 
colon adenocarcinoma when cultured in vitro in agar did not show contact 
inhibition. 
The methods described above were used for developing cultures of primary 
tumor cells of human colon adenocarcinoma in vitro and for monitoring the 
cells' viability, true identity, growth, and tumorigenicity. A success 
rate of 100% (12/12) was achieved in establishing the primary tumor cell 
cultures. A success rate of 100% was considered achieved when all twelve 
cell lines established were passaged at least 5 times in vitro. 
In Vivo Studies 
The primary tumor cells of human colon adenocarcinoma that were cultured in 
vitro and identified as adenocarcinoma cells by the monoclonal antibody 
probe were used for tumor induction in nude mice. Six-to-eight-week-old 
nude mice of NIH strain (nu/nu) of both sexes were selected for tumor 
induction and were maintained in a sterile environment. Three mice per 
cell line were used. All the animals selected were in good health as 
indicated by their stable weight and normal activity. All the animals were 
maintained on a daily cycle of 12 hours light and 12 hours dark. The 
animals were fed with an autoclaved standard pellet diet and water ad 
libitum. Sterilized cages were changed weekly. For standardization of the 
technique, various sites of injection and tumor cell concentrations 
optimum for tumor formation were tried. Tumor cell concentrations ranging 
from 10.sup.6 cells/0.2 ml to 2.times.10.sup.7 cells /0.5 ml were injected 
in the nude mice. The cells came from a 4-to-5-day-old culture from an 
established cell line that had undergone at least 5 passages. Injection 
sites were selected from the peritoneum, subcutaneously on the back of the 
animal, and subcutaneously on the abdomen. Each mouse was injected in one 
of these three sites. It was found that injection subcutaneously on the 
abdomen was optimum. The growth kinetics of the abdominal tumors were 
monitored by measuring the dimensions of the tumor using a vernier 
calliper, from which the volume was mathematically derived (Winn, 1959). 
The abdominal tumor was photographed every third day, and the weight was 
determined after excision of the tumor. 
It was found that a primary tumor cell concentration of 10.sup.7 cells/0.3 
ml of the tumor cell suspension of primary tumor cells of human colon 
adenocarcinoma was optimum. Cell numbers less than approximately 10.sup.7 
did not result in tumor formation. Similarly, cells injected into the back 
or the peritoneum of the animal did not result in the formation of a 
visible tumor mass. 
In another experiment, a primary tumor cell concentration of 10.sup.7 
cells/0.3 ml of the tumor cell suspension was injected subcutaneously on 
the abdomen in 24 nude mice. A palpable mass at the site of the tumor cell 
injection appeared within 3 to 4 days post-injection (day 0 is the day when 
the mice were injected with the cells), and the mass subsequently began to 
grow as a solid tumor. The mean tumor volume was about 100 mm.sup.3 by day 
12 and increased to approximately 300 mm.sup.3 by day 16. A phase of rapid 
growth was observed from day 16 to 28, at which time the volume increased 
to about 1500 mm.sup.3. From day 28 to 32, there was a very rapid growth 
of the tumor and a volume of approximately 2400 mm.sup.3 was attained. The 
tumor volume rarely increased beyond 3000 mm.sup.3. A necrotic spot 
appeared in all of the tumors in all of the mice. The necrotic spot was 
about 1 mm diameter and appeared in the center of the tumor after about 
days 14-16, and the spot increased in size with time. FIG. 1 shows the 
mean tumor volumes. 
All of the mice died between day 28 and day 32. 
Additional In Vitro and In Vivo Studies 
Cells from the tumor tissue growing in the nude mice then were cultured in 
vitro. For this, a tumor-bearing mouse was sacrificed on day 25. The tumor 
was excised; and a single-cell suspension of tumor cells from the tumor was 
prepared in a manner identical to the one described earlier for human 
primary tumors. Cultures of primary tumor cells of human colon 
adenocarcinoma were reestablished from the excised tumor of the nude 
mouse. The cultured cells retained adenocarcinoma characteristics as 
revealed by testing with the monoclonal antibody probe. The cells could be 
passaged several times in vitro. The cultured tumor cells also retained 
their ability to form colonies in soft agar, thus indicating that the 
cells retained their tumorigenic properties. The cultured tumor cells were 
reinjected into nude mice to check the tumor growth in vivo. A cell 
suspension having a concentration of 10.sup.7 cells/0.3 ml was prepared 
and injected subcutaneously on the abdomen of the nude mouse. The tumor 
growth kinetics were similar to the growth kinetics obtained when 
established cell line from primary tissue was injected at same 
concentration and route. 
This specification incorporates herein the following article by this 
reference: Jaggi, M., Mukherjee, R., "Establishment of Tumorigenic Cell 
Lines from Biopsies of Human Colon Adenocarcinomas," Journal of Basic & 
Applied Biomedicine, 3(4):27-35 (1995). 
The invention illustratively disclosed herein suitably may be practiced in 
the absence of any element which is not specifically disclosed herein. 
Thus, the invention may comprise, consist of, or consist essentially of 
the elements disclosed herein. 
Although the present invention has been described in considerable detail 
with reference to certain preferred embodiments, the spirit and the scope 
of the appended claims should not be limited to the description of the 
preferred embodiments contained herein. Thus, although a few exemplary 
embodiments of this invention have been described in detail above, those 
skilled in the art will readily appreciate that many modifications are 
possible in the exemplary embodiments without materially departing from 
the novel teachings and advantages of this invention. Accordingly, all 
such modifications are intended to be included within the scope of this 
invention as defined in the followings claims. 
The following claims are entitled to the broadest possible scope consistent 
with this application.