KR100721927B1 - How to separate cancer cells from cancer tissue - Google Patents

Abstract

The present invention relates to a method for separating cancer cells from cancer tissue, and more particularly, pretreatment of the isolated cancer tissue with alcohol and washing with a washing solution containing antibiotics; Separating the cancer cells by floating the washed cancer tissues in an enzyme mixture containing antibiotics; And it relates to a method for separating cancer cells comprising the step of removing non-cancer cells from the cell suspension.

Cancer Cell Separation, Solid Cancer

Description

A method of separating tumor cells from cancer tissue

Figure 1 shows the trypan blue staining of the cell suspension immediately after the separation of solid cancer tissue, Figure 1b is an enlarged view of the circle shown in the dotted line of Figure 1a, where L represents a living cell (living cell) Unstained, D stained to represent dead cells, blue in color, and R to red blood cells, not stained, highly refractive.

Figure 2 shows the result of HE staining (Haematoxylin & Eosin Staining) immediately after the separation of solid cancer tissue.

Figure 3 shows the distribution of cancer cells and non-cancer cells present in solid cancer tissue as a result of Ficol Gradient Centrifuge for solid cancer tissue.

Figure 4 shows the result of removing the non-cancer cells from the solid cancer tissue as a result of immunomagnetic separation (immunomagnetic separation) for the solid cancer tissue.

FIG. 5 shows cancer cell separation efficiency according to carcinomas of five major carcinomas of Korea (stomach cancer, lung cancer, liver cancer, colon cancer, breast cancer). FIG. 5A shows the cancer cell yield and FIG. 5B shows the survival rate of the obtained cancer cells.

Figure 6 shows the result of the culture success rate of the colon cancer tissue having low culture success rate compared to the general solid cancer increased after the treatment process according to the present invention. 'Average' is the culture success rate of solid cancers other than colon cancer, and 'Colour cancer (before treatment)' is the culture success rate when only washing solution containing antibiotics is used. When used, it means the culture success rate.

In Korea, about 9,000 new cancer patients occur each year (2002 Korea Central Cancer Registration Project). According to the recent 10 years of cancer registration, the average annual cancer incidence increases by about 5.6% per year, and as of 2001, the cancer mortality rate per 100,000 population was 238.8 in Japan and 200.5 in the United States, compared to 123.5 in Korea. In view of the fact that the cancer cases in Korea is expected to continue to increase in the future.

With the recent development of life science technology, attempts have been made to utilize human cancer tissues and cancer cells isolated therefrom for diagnosis, treatment, prognosis, or basic cancer research. However, since various various cells are incorporated in cancer tissues, it is difficult to separate only pure cancer cells, and separation of a sufficient amount of living cancer cells from various solid cancer tissues may be a technical obstacle. Researchers in the United States are using laser microseparation to separate cancer cells one by one from living cancer tissues, but until now, there is no such technology in Korea, and cancer cells are cultured and studied in a laboratory.

In the case of isolating live cancer cells from solid cancer tissue, subsequent studies may require cell culture processes such as cancer cell line formation, chemosensitivity testing, and cancer vaccine development. Therefore, in order to efficiently isolate cancer cells and use them for further cell culture, the process of inhibiting microbial contamination by the fungi present in human tissues, separating the living cells sufficiently, and removing normal cells incorporated need.

Therefore, the present inventors have devised a method to remove normal cells incorporated from the five major carcinomas of Koreans, stomach cancer, lung cancer, liver cancer, colorectal / rectal cancer, and breast cancer tissues, and increase the proportion of cancer cells in the sample, and prevent microbial contamination. The invention of the pretreatment process and the use of effective antibiotics to increase the culture efficiency of colorectal, rectal cancer cells, which are difficult to do, was completed.

Accordingly, an object of the present invention is to reduce the microbial contamination of cancer cells, and to efficiently remove non-cancer cells from solid cancer tissues to provide a method for separating cancer cells with high cancer cell separation efficiency.

In order to achieve the above object, the present invention is an aspect,

(a) pretreating the separated solid cancer tissue with alcohol and washing with a wash solution comprising antibiotics; (b) floating the washed cancer tissue in an enzyme mixture including penicillin, streptomycin, gentamicin and empoterisin B to separate cancer cells; And (c) provides a method for separating solid cancer cancer cells comprising the step of removing the non-cancer cells from the cell suspension. In a more preferred embodiment, the method provides a method for separating solid cancer cancer cells, characterized in that it further comprises matronidazole in the enzyme mixture of step (b).

In the present invention, the term "solid cancer" refers to an abnormal growth of cells in a solid organ such as the breast or the prostate, as opposed to leukemia, which affects liquid blood. Therefore, the solid cancer of the present invention includes gastric cancer, lung cancer, liver cancer, colon cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer, kidney cancer, melanoma, small cell and non-small cell lung cancer, sarcoma, neuroblastoma Glioma or T-cell lymphoma and B-cell lymphoma, and the like.

'Pretreatment of the solid cancer tissue isolated in the method of the present invention with alcohol' preferably includes disinfecting the tissue surface by immersing the cancer tissue in 70% ethanol. The conventional cancer cell separation method does not include a process of pretreatment with alcohol or the like before washing the separated cancer tissues with a washing buffer containing antibiotics. As demonstrated in the following Examples, by the pre-treatment with alcohol before washing with a washing solution containing antibiotics as in the method of the present invention, to prevent contamination in the culture process of isolated cancer cells to improve the purification rate and yield of cancer cells The height had a remarkable effect.

The 'washing liquid' containing the antibiotic may be a washing liquid generally used in conventional cancer cell separation, but preferably, the antibiotics of the same type as those included in the enzyme mixture according to the present invention to be described later, penicillin It may be a solution containing antibiotics, such as streptomycin and gentamicin, further comprising fetal calf serum (FBS). In a preferred embodiment of the present invention, 100 U / ml penicillin, 100 µg / ml Streptomycin, 100 ug / in an antibiotic solution containing Empoterisin B (HBSS, Hank's balance salt solution, Hyclone) A solution containing ml gentamicin, 2.5 μg / ml empotericin B and 10% FBS was used as the wash in step (a).

In the present invention, the "enzyme mixture" refers to a solution containing an enzyme capable of separating cancer cells and non-cancer cells from a solid cancer tissue isolated from a human body having cancer and a substance capable of increasing other separation efficiency. Preferably, enzymes capable of separating cancer cells from non-cancer cells in the enzyme mixture include collagenase, dispase, pronase, DNase, etc. (enzyme composition reference: Sevin BU , Peng ZL, Perras JP, Ganjei P, Penalver M, Averette HE.Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing.Gynecol Oncol. (1988), 31, 191-204), Antibiotics such as penicillin, streptomycin, gentamicin, ampotericin B, and metronidazole are used to prevent cancer cell contamination. It may be a culture medium such as PMI 1640 containing FBS as a substance. RPMI 1640 culture medium used in the separation enzyme mixture can be easily replaced with a culture medium such as DMEM, RPMI, IMDM, MEM, McCoy's 5A, but is not limited thereto.

In a specific embodiment of the present invention, the enzyme mixture is RPMI 1640, collagenase, collagenase, pronase, DNase, penicillin, streptomycin, gentamicin, gentamicin, empotericin B (Ampotericin B), metronidazole (Metronidazole) and consisting of a culture medium containing FBS, except for the case of colorectal cancer and rectal cancer, cancer cells can be separated with excellent efficiency even if the component does not contain metronidazole. In the case of colorectal cancer and colorectal cancer, the purification efficiency of cancer cells is much higher and the success rate of culture is increased especially when metronidazole is included.

Each component and concentration of the enzyme mixture can be appropriately adjusted by those skilled in the art as needed, and reference can be made to well-known literature on methods for separating cancer cells from non-cancer cells using such enzyme mixtures. 40: 4-7, 1989; Cancer research 43, 258-264, 1983: Amphotericin 1ug / mi; and Cancer research 37, 3639-3643, 1977 & Eur. J Cancer 33 (8) 1291-1298, 1997). Among the antibiotics used in the invention, penicillin is Gram-positive bacteria, Streptomycin is Gram-positive and Gram-negative bacteria, Gentamicin is Gram-positive and Gram-negative bacteria and mycoplasma, and Empotericin B has antimicrobial effects against fungi and yeast. These antibiotics can be replaced with other antibiotics having the same range of action.

Specifically, other antibiotics that can act on gram-positive and gram-negative bacteria include neomycin and ampiciliin, and other antibiotics that can act on gram-positive and gram-negative bacteria and mycoplasma. Kanamycin is an antibiotic that can act on fungi and yeast, and Nystatin (Culture of animal cells, WILEY-LISS, 4TH edition, 99 pages).

When the solid cancer tissue is cut with a surgical knife and suspended in the enzyme mixture, the cancer cells and the non-cancer cells are separated from the cancer tissue, and the non-cancer cells can be separated from the cell suspension containing the separated cancer cells and the non-cancer cells. . In the present invention, the term "non-cancer cell" means all cells except cancer cells in solid cancer tissues, and includes blood cells and other normal cells. Treatment with the enzyme mixture causes the cancer tissue to float in the enzyme mixture, approximately 37 ° C., 5% CO _2. Incubating for 12 to 16 hours depending on the type of cancer tissue in the presence.

Removing the non-cancerous cells from the cell suspension treated with the enzyme mixture includes using Ficoll gradient centrifugation and anti-CD45 antibody magnetic beads.

The "picol density gradient centrifugation" is a centrifugation method using a density gradient of a picol, a synthetic sucrose polymer, and cells having different densities can be separated during the precipitation process according to the concentration of the picol present in the solution. (Katayanagi N. et al. “Flow cytometric BrdU / DNA assay for anticancer agent sensitivity test” Nippon Rinsho. 1992, 50 (10), 2386-2390; Silva SL et al. “A novel model for isolation of Walker's tumoral cells using the Ficoll-Hypaque gradient ”Acta Cir Bras. 2006, 21 (2), 101-105; Weisethal LM, et al.“ A novel dye exclusion method for testing in vitro chemosensitivity of human tumors ”Cancer Res. 1983, 43 (2), 749-57; and Andreotti PE et al. “Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma” Cancer Res. 1995, 55 (22), 5276-5282 . Reference). In the present invention it was confirmed that by using this for cancer cell separation can significantly increase the efficiency of separating cancer cells and non-cancer cells in cancer tissue.

The "anti-CD45 antibody magnetic beads" is a magnetic bead method using an anti-CD45 antibody, wherein the magnetic beads are a uniform superparamagnetic coated with silica, having a diameter of 1 to 5, and an amine. , Beads with other surface groups such as Carboxy, Aldehyde, Epoxy, IDA, hydrazide, DADPA or Silica. These beads are widely used to separate proteins, antibodies, carbohydrates, lectins, nucleic acids, or small drugs due to their low nonspecific binding rate, wide surface area, good dispersion, and easy handling.

In a particularly preferred embodiment, the present invention provides a method for separating cancer cells, characterized in that the cancer cells are separated from the tissue of colorectal cancer or colorectal cancer in solid cancer with high purification rate and yield. When the cancer cells are separated from the colon and rectal cancer tissues, they tend to be easily contaminated by residing fungi present in the human body, compared to other solid cancers, and thus, there is a difficulty in separating and culturing. In the present invention, particularly in the alcohol pretreatment and the enzyme mixture as described above. By further including metronidazole, it was confirmed that cancer cells can be cultured from colorectal cancer or rectal cancer with purification efficiency and yield of general solid cancer levels.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only intended to illustrate the contents of the present invention, it should not be construed that the scope of rights is limited thereto.

< Example >

Tissues of gastric cancer, lung cancer, liver cancer, colorectal cancer, and breast cancer, the five most common solid carcinomas obtained from Severance Hospital (Yonsei University College of Medicine), were treated in the same manner according to the following examples. Rather than sorting the tissues by type, they are all described in the term “solid cancer”.

< Example  1> Pretreatment of Cancer Tissue

The solid cancer tissue was soaked in 70% alcohol (ethanol, Sigma) for 2-3 seconds to disinfect the tissue surface, and immediately after the surface disinfection, it was transferred to a petri dish and poured 20 ml of washing media A and wiped out the tissue well. Washing solution A was prepared by HBSS (Hank's balance salt solution, Hyclone) in 100 U / ml penicillin, 100 μg / ml streptomycin, 100 ug / ml gentamicin, 2.5 μg / ml empotericin B And a solution containing 10% FBS (Ref .: Sevin BU, Peng ZL, Perras JP, Ganjei P, Penalver M, Averette HE.Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing.Gynecol Oncol. ( 1988), 31, 191-204).

Remove used wash solution and wipe again with fresh wash solution A. The tissues were cut to the extent of 1.5ml tubes and weighed into 1.5ml tubes. The tissues were transferred back to Petri dishes and the empty 1.5 ml tubes were weighed. The tissue was transferred to a clean Petri dish and chopped with a surgical knife or surgical scissors to less than 1 mm ² .

< Example  2> Separation enzyme mixture solution treatment

The solid cancer tissue pretreated as in Example 1 was evenly distributed in 5-10 ml of a disaggregation enzyme cocktail, and then reacted for 12 hours depending on the type of cancer tissue in the incubator at 37 ° C. 5% CO ₂ . I was. The separation enzyme mixture is cultured in RPMI 1640, collagenase II (collagenase II), pronase, DNase, penicillin, streptomycin (Streptomycin), empoterisin B (Ampotericin B), gentamicin (gentamicin), metronidazole, and a solution containing 10% FBS were used.

The concentration of enzymes actually used in the present invention is 0.2mg / ml collagenase II, 2.25 PUK / ml pronase, 1400 U / ml Dnase (Reference: Sevin BU, Peng ZL, Perras JP, Ganjei P, Penalver M, Averette HE. of an ATP-bioluminescence assay in human tumor chemosensitivity testing.Gynecol Oncol. (1988), 31, 191-204, and the actual concentration of antibiotics used was 100 U / ml penicillin, 100 µg / ml streptomycin. , 2.5 μg / ml Empotericin B, 100 μg / ml gentamicin 1 μg / ml metronidazole.

< Example  3> Cancer Cell Recovery

Cancer tissue treated with the enzyme mixture in Example 2 was pipetted 20 times with a 10 ml serological pipette so that the cells inside the tissue sections could be well separated. When the cancer tissue to be decomposed was small and a small amount of separation enzyme mixture was used, about 5 ml of washing solution A was added and pipetted. Cell suspensions were filtered through strainers into 50 ml conical tubes. Residual cells present in traces in Petri dishes and filters were recovered by resuspending with fresh Wash A. The filtrate was centrifuged at 500 g for 5 minutes to obtain pellets. The supernatant was removed with a 10 ml serum pipette, and the pellet was again suspended in 1 ml of washing solution A, transferred to a 1.5 ml tube, and centrifuged at 500 g for 5 minutes to obtain pellets. The pellet was resuspended in a suitable volume (200-100 μl) of Wash Solution A, which was then trypan blue stained and cell counted.

Immediately after cancer tissue was isolated, the most common non-cancerous cells were red blood cells. The results are stained with trypan blue for cell suspension prior to separation Ficoll density gradient centrifugation showed in Figure 1. As shown in FIG. 1B, L represents living cells and is not stained, D is stained to represent dead cells and is blue in color, R represents red blood cells and is not stained, very Highly refractive. Numerous erythrocytes and fewer nucleated cells were observed and dead cells stained with trypan blue.

< Example  4> Removal of red blood cells and dead cells

2 ml of Ficoll (Sigma H4153, density = 1.077 g / ml) was placed in a 15 ml tube, and 3 ml of the cell suspension prepared thereon was carefully flowed along the tube wall using a paste pipette or P1000 pipetteman. . At this time, about 8 × 10 ⁶ cells are suitable for one tube, and if it is more than that, the number of tubes is increased, divided, and centrifuged at 400 g for 15 minutes at room temperature. Carefully take the interface with a Pipetteman or Passer pipette and transfer it to a 15 ml tube, and take small portions above and below the interface of Wash Solution A and Piccol to increase yield. The tube was filled with washing solution A, shaken, washed, and centrifuged at 500 g for 5 minutes. The pellet was resuspended in 1 ml of wash solution, transferred to a 1.5 ml tube, and centrifuged at 500 g for 5 minutes. The pellet was resuspended in a suitable volume (200-1000 μl) of Wash Solution A, stained with trypan blue, and counted.

Ficoll density gradient centrifugation (1.077 g / ml) can be used to remove more than 95% of the erythrocytes incorporated from the nucleated cell layer, which also helps to remove cell debris and dead cells from pretreatment for culture. .

< Example  5> Noncancerous  remove

In order to investigate the ratio and distribution of cancer cells and non-cancer cells in cancer tissues, gastric cancer tissues belonging to solid cancer tissues were isolated and cytosine (thermosindon) slides were prepared using cell suspension and HE staining (Haematoxylin & Eosin Staining) Was implemented. The stained nucleated cells were read by a pathologist to determine whether they are cancer cells, non-cancer cells, cell types, etc., and the size of the cells was classified using Image Pro. As a result, 58% of all cells were non-cancerous cells, and the most common non-cancerous cells in cancer tissues were red blood cells, lymphocytes, macrophages, difficult to classify cells, neutrophils, and fibroblasts ( Fig. 2 ) . ).

Wash cells once with washing buffer B (solution containing 1 × Phosphate-Buffered Saline pH7.4 (Gibco), 0.5% BSA and 2 mM EDTA) and re-use with 80 μl of wash buffer per 10 ⁷ cells. Suspended and 80 μl of Wash B was used for up to 10 ⁷ cells. 20 μl of MACS anti-CD45 beads per 80 μl of cell suspension were added and mixed well, followed by incubation at 6-12 ° C. for 15 minutes. Incubation was placed in the refrigerator 1-2 times (inversion). Washing solution B corresponding to 10 to 20 times the total volume of the mixture (mixture) was added and washed, and centrifugation was performed at 300 g for 10 minutes to remove unbound beads present in the supernatant. After centrifugation the supernatant was removed and the pellet was resuspended in 500 μl wash buffer. The MACS Multistand was sterilized with alcohol and placed in a clean bench, and the OctoMACS separator was also sterilized and attached to the multistand. Under the separator, a rack and an appropriate volume of 1.5 ml tube were prepared to receive the fragments coming out of the column. After inserting the MS column into the separator, the washing buffer was prewetting while flowing about 500 μl. Thereafter, the cell suspension prepared above was flowed to the column. When the cell suspension was drained out, Negative selection of cells with no beads bound in the column was performed by flowing Wash Solution B about 500 μl three times, and all the fragments that were negatively selected were collected and centrifuged at 500 g to obtain pellets. . The pellet was resuspended in a suitable volume (200-1000 μl) of Wash Solution A, stained with Trypan Blue, and counted.

Ficoll density gradient centrifugation allowed the separation of polymorphonuclear leukocytes from mononuclear cell layers. As a result of examining the percentage of cancer cells before and after Ficoll density gradient centrifugation in surgically extracted solid cancer tissues, it was observed that the proportion of cancer cells increased from 40.9% to 45.6% on average ( FIG. 3 ). Commonly incorporated normal cells were erythrocytes, white blood cells, macrophages, difficult to sort normal cells, neutrophils, and fibroblasts.

Since most of the normal cells incorporated into cancer tissues are blood-derived cells, the products using magnetic beads combined with antibodies to CD45 expressed on the surface of nucleated blood are used. Negative selection of the cells resulted in an average increase of 21.7% in cancer cells in 18 solid cancer tissues. However, when the percentage of cancer cells in the tissues was low (less than 20%), the rate of increase of cancer cells was slight even after Ficol density gradient centrifugation and immunomagnetic separation ( FIG. 4 ). This means that the stage at which cancer tissue is collected is very important.

As a result of the above example, the separation efficiency of each type of cancer among the five most common carcinomas of Korea, as shown in Figure 5a, 50,000 cells per mg in gastric cancer (n = 129), mg in lung cancer (n = 67) About 38,000 cells per mg, about 49000 cells per mg for liver cancer (n = 15), about 31000 cells per mg for colorectal cancer (n = 26), and about 15000 cells per mg for breast cancer (n = 75) As shown in FIG. 5B, the survival rate of these isolated cancer cells was 92.2% (n = 129), lung cancer 94.9% (n = 67), liver cancer 93.7% (n = 15), and colon cancer 91.9%. (n = 26), and 92.4% (n = 75) of breast cancer, indicating a significantly improved separation efficiency (yield) and survival rate of the isolated cells compared to the conventional method.

In addition, the increase in the success rate of culture in colorectal cancer tissue compared to other solid cancer is shown in FIG . Using a conventional cancer cell culture method, the success rate of the culture of cancer cells separated according to the method of the present invention and cancer cells separated by the conventional method was compared, the success rate of culture of solid cancer cells other than colorectal cancer isolated according to the method of the present invention Was 95%. In addition, the culture success rate of the colon cancer cells isolated according to the present Example including the ethanol pretreatment was 57%, whereas the culture success rate of the conventional colon cancer cell separation using only the washing solution containing antibiotics was 94%, the present invention In the case of separating cancer cells according to the method, it is difficult to expect high purification rate due to its characteristics, such as colorectal cancer or rectal cancer, in addition to general solid cancer, and thus high separation and purification efficiency in cancer tissues that could not be expected to have high culture success rate. It can be seen that the subsequent high cultivation success rate can be guaranteed.

As described above, the cancer cell separation method of the present invention comprises the steps of pretreating the separated solid cancer tissue with alcohol and washing with a washing solution containing antibiotics; Separating the cancer cells by floating the washed cancer tissues in an enzyme mixture containing antibiotics; And removing the non-cancer cells from the cell suspension, the cancer cell separation yield is superior to the existing methods, and the contamination inhibition rate in the culture of the isolated cancer cells is high, and cancer diagnosis, treatment, prognosis and basic diagnosis of cancer patients It can be widely used for cancer research, which is particularly effective in the case of colorectal cancer or rectal cancer, which had not been expected to have high purification rate and yield rate due to the characteristics of cancer tissue.

Claims (9)

Pretreatment of the separated solid cancer tissue with alcohol and washing with a washing solution containing antibiotics; Separating the cancer cells by floating the washed cancer tissues in an enzyme mixture including penicillin, streptomycin, gentamicin and empoterisin B; And removing blood cells and normal cells from the cell suspension. The method of claim 1, wherein the enzyme mixture further comprises metronidazole. The method of claim 1, wherein the pretreatment of the cancer tissue with alcohol comprises disinfecting the tissue surface by immersing the cancer tissue in 70% ethanol. The method of claim 1, wherein the enzyme mixture is collagenase II (collagenase II), dispase, pronase, DNase, penicillin (penicillin), streptomycin, gentamicin (gentamicin) in the culture medium ), Empotericin B (Ampotericin B), Metronidazole (Metronidazole) and FBS solution containing cancer cells separation method. The method of claim 4, wherein the enzyme mixture is 0.05 to 0.8 mg / ml collagenase II (collagenase II), 1 to 5 PUK / ml pronase, 600 to 2000 U / ml DNase, 50 to 200 U / ml penicillin (penicillin), 50 to 200 μg / ml streptomycin, 50 to 200 ug / ml gentamicin, 0.5 to 5 μg / ml empotericin B, 1 μg / ml metronidazole ) And a method for separating cancer cells containing 10% FBS. 6. The method of claim 4 or 5, wherein the culture is one or more cultures selected from the group consisting of DMEM, RPMI, IMDM, MEM and McCoy's 5A. The method of claim 1, wherein the step of removing blood cells and normal cells cancer cell separation, characterized in that using Ficoll gradient centrifugation and anti-CD45 antibody magnetic beads (Anti-CD45 antibody magnetic bead) Way. The method according to any one of claims 1 to 5 and 7, wherein the cancer tissue to be treated is gastric cancer, lung cancer, liver cancer, colon cancer, rectal cancer, breast cancer, prostate cancer, skin cancer, head and neck cancer, pancreatic cancer, ovarian cancer, bladder cancer A method for isolating cancer cells that are renal cancer, melanoma, small cell and non-small cell lung cancer, sarcoma, glioma or T-cell lymphoma and B-cell lymphoma tissue. The method of any one of claims 2 to 5 and 7, wherein the cancer tissue to be treated is colorectal cancer or rectal cancer tissue.

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