CN109609463B - Separation and culture method of primary cervical cancer tumor cells - Google Patents
Separation and culture method of primary cervical cancer tumor cells Download PDFInfo
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- CN109609463B CN109609463B CN201910021776.4A CN201910021776A CN109609463B CN 109609463 B CN109609463 B CN 109609463B CN 201910021776 A CN201910021776 A CN 201910021776A CN 109609463 B CN109609463 B CN 109609463B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a separation and culture method of primary cells of cervical carcinoma tumors, which comprises the following steps: 1) placing the tissue blocks into a precooled sterile culture solution for treatment; 2) tissue cleaning and tissue cutting; 3) digestion treatment; 4) filtering with sterile cell filter to obtain digestive filtrate, and standing at low temperature; 5) washing the sterile cell filter by collagenase, and collecting the sterile cell filter into a centrifuge tube; 6) horizontally placing the centrifugal tube in a vibration box for fully vibrating; 7) combining digestive juice obtained in the steps 4) and 6), centrifuging, and removing supernate; 8) fully blowing and beating the heavy suspension cell sediment; 9) separating and passaging; 10) and (4) magnetic bead sorting. The invention has the beneficial effects that: can prolong the tissue in vitro time, improve cell growth efficiency, shorten the time cycle, the purification is efficient, provides novel high-efficient scheme for cervical carcinoma basic research.
Description
Technical Field
The invention relates to the field of modern biotechnology, in particular to a separation and culture method of primary cells of cervical cancer tumors.
Background
Worldwide, Cervical Cancer (CC) is the female malignancy second only to the incidence of breast cancer, and it is well known that persistent infection of HPV at high risk is the causative factor, and although it has been shown in clinical practice of HPV vaccine popularization for ten years that the incidence of CIN ≧ III is reduced by at least 50% (American clinical tumor Association report, 2016), cervical cancer remains the most fatal disease of female reproductive system tumor due to the differences in large-scale screening and primary prevention dissemination, together with the lack of effective HPV + resolution strategies. The Chinese national Cancer center written article (Cancer statistics in China, 2015) shows that the incidence of cervical Cancer reaches 99/100, 000 people and 31/100, 000 people die, seriously threatens the physical and mental health of women in China, and still has great significance for the research of the pathogenesis of the disease, so the establishment of a research model is the research basis.
Currently, cervical cancer cell lines include Hela (cervical adenocarcinoma derived) and SiHa, CaSki, C4-1 and C33A, which are widely used, and have fewer types than other cancer cell lines, and thus cannot sufficiently satisfy the research requirements. The primary cell culture is used as an excellent experimental model and is researched by researchers in the last 30 years, however, the cervix is in a natural cavity of a human body and is in a non-sterile environment, and meanwhile, a cervical cancer patient is mostly infected with a focus, various flora exist, and cervical tumor tissues contain various cell components, such as cancer cells, cancer-related fibroblasts, tumor-infiltrating lymphocytes, vascular endothelial cells and blood cells, so that a research barrier is created for the success rate of primary cell isolation culture and the purification of the tumor cells.
An effective solution to the problems in the related art has not been proposed yet.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a separation and culture method of primary cervical carcinoma tumor cells, which can prolong the tissue in vitro time, improve the cell growth efficiency, shorten the time period, have high purification efficiency and provide a novel efficient scheme for the basic research of cervical carcinoma.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a separation and culture method of primary cervical cancer tumor cells comprises the following steps:
1) placing the cut tissue block into a precooled culture solution, wherein the diameter of the tissue block reaches 0.8 cm, placing the tissue block at a low temperature, and transporting the tissue to a laboratory in an aseptic, low-temperature and nutrient-rich environment, so as to prolong the processing time of the tissue and strive for time for tissue sampling and experimental preparation work;
2) lightly wiping blood of the tissue block with gauze in a super clean bench and washing, placing the cleaned tissue block into a 6cm culture dish added with 0.5mL culture solution, cutting the tissue into 1mm multiplied by 1mm in the culture solution, ensuring that the tissue keeps a nutrient and moist state in the time of cutting the tissue block, and determining the digestion degree according to the size of the cut tissue;
3) adding 2ml of digestive juice into the broken block tissues in the step 2), placing the mixture into an incubator for digestion treatment to obtain the digestive juice containing cell suspension, observing the dissociation condition of the tissues, and if the fibriform content is high and the tissue blocks are hard, prolonging the digestion time and stopping the reaction if the tissues are sticky;
4) filtering the digestive juice containing the cell suspension in the step 3) to a 50mL test tube filled with 2mL fetal calf serum by using a 100um sterile cell filter, washing the digestive juice with PBS to obtain digestive filtrate containing the fetal calf serum, and placing the digestive filtrate at a low temperature to enable the cells to be in a low-temperature environment;
5) reversely buckling the 100um sterile cell filter in the step 4) on an original culture dish, washing the filter screen of the 100um sterile cell filter by using 0.2% I-type collagenase, completely washing the viscous tissue on the filter screen into the culture dish, totally using 6mL of 0.2% I-type collagenase, transferring the washing liquid to a 15mL centrifuge tube, and further digesting the undigested tissue transferred centrifuge tube;
6) flatly placing the centrifuge tube in the step 5) in a shaking box for sufficient shaking, so that the residual tissue is sufficiently digested, and obtaining a tissue fragment digestive juice;
7) adding the digestion filtrate containing fetal calf serum in the step 4) into the tissue fragment digestion solution in the step 6), centrifuging, and removing supernatant to obtain cell sediment;
8) fully blowing and beating the resuspended cell sediment by using culture solution, counting by using a cell counting plate, and transferring the obtained cells into an incubator for culture;
9) separating and passaging, wherein the cells present cobblestone appearance;
10) the cells were subjected to EpCAM/CD326 magnetic bead sorting, CD326+ being pure cervical carcinoma squamous epithelial carcinoma cells, CD 326-containing cervical squamous epithelial carcinoma cells and a small number of fibroblasts.
Further, the culture solution in the step 1), the step 2) and the step 8) is one of DMEM high-glucose culture solution +5% FBS +1% antibiotic + HEPES 10mM/mL, DMEM/F12 +10% FBS +1% antibiotic or DMEM-high-glucose culture solution.
Further, the low temperature in the step 2) is stored in a refrigerator or an ice bag at 4 ℃.
Further, the clean-up in step 2) was washed with PBS +10% antibiotics.
Further, in the step 3), the digestion solution is 0.05% trypsin containing EDTA, and the digestion time is 15-30 min.
Further, the culture conditions of the culture boxes in the step 3) and the step 8) are 5% CO2、37℃。
Further, the temperature of the oscillating box in the step 6) is 37 ℃, the rotating speed is 180r/min, and the oscillating time is 45-60 min.
Further, the magnetic beads are sorted to EpCAM/CD326 positive tumor cells in the step 10).
The invention has the beneficial effects that: placing the cut tissue blocks into a precooled sterile culture solution, and placing at a low temperature, so that the in-vitro time of the tissue can be prolonged; washing a filter screen of the sterile cell filter, and fully digesting to extract a large number of cervical cancer tumor primary cells with high survival rate; the extracted primary cell has high cell growth efficiency, short time period and high purification efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of a process of isolating and culturing primary cells of cervical squamous carcinoma tissue according to example 1 of the present invention;
FIG. 2 is a schematic diagram of magnetic bead sorting of a cervical squamous carcinoma tissue cell specimen according to embodiment 1 of the present invention;
FIG. 3 is a graph showing HE staining results of cervical squamous carcinoma tissue according to example 1 of the present invention;
FIG. 4 is a graph showing the result of measuring the ratio of components in the cervical squamous carcinoma tissue according to embodiment 1 of the present invention;
FIG. 5 is a graph showing the result of culture observation of the separation of the cell specimen of cervical squamous carcinoma tissue according to example 1 of the present invention;
FIG. 6 is a graph showing the result of magnetic bead sorting and culturing of a cervical squamous carcinoma tissue cell specimen according to example 1 of the present invention;
FIG. 7 is a graph showing the results of expression of AE1+ alpha-SMA, AE1+ P16, P16+ HPV16/18-E6 in the cervical squamous cell carcinoma specimen according to example 1 of the present invention. FIG. 7A shows the expression of AE1+ alpha-SMA in a cervical squamous carcinoma cell specimen; FIG. 7B shows the expression of AE1+ P16 in cytology specimen of cervical squamous carcinoma; FIG. 7C shows the expression of cervical squamous carcinoma tissue cell specimen P16+ HPV 16/18-E6;
FIG. 8 is a graph showing the result of the magnetic bead sorting EpCAM/CD326+ cell purity detection of the cervical squamous carcinoma tissue cell specimen according to embodiment 1 of the present invention;
FIG. 9 is a statistical graph of the detection expression ratios of multiple groups of EpCAM/CD326+ cell cancer stem cell markers sorted by magnetic beads according to the method described in example 1 of the present invention;
FIG. 10 is a graph of the results of magnetic bead sorting EpCAM/CD 326-multicolor flow cytometric marker analysis according to example 1 of the present invention;
FIG. 11 is a statistical graph of the detection expression ratios of sets of EpCAM/CD 326-fibroblast (Vimentin +) markers sorted from magnetic beads according to the method described in example 1 of the present invention;
FIG. 12 is a graph showing the result of 14d cell clone formation in a cervical squamous carcinoma tissue cell specimen according to example 1 of the present invention;
FIG. 13 is a graph showing the results of comparison of the sorting of EpCAM/CD326+, EpCAM/CD 326-and unsorted cells into a cervical cancer tissue isolation cell position doubling time according to example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
1) Placing the excised tissue block into culture solution which is pre-cooled and is placed with DMEM/F12 +10% fetal bovine serum +1% antibiotics, storing at 4 ℃, wherein the HE staining detection result of cervical squamous carcinoma tissues is shown in figure 3, wherein A, B, C and D are HE staining of cervical squamous carcinoma tissues, E is mouse anti-human AE1/AE3 antibody staining (10X), I is mouse anti-human P63 antibody staining (10X), G is mouse anti-human P16 antibody staining (10X), H is mouse anti-human Ki67 antibody staining (10X), and is definite cervical squamous carcinoma, the proportion result of cervical carcinoma tissue components is shown in figure 4, the cervical carcinoma tissues are mouse anti-human AE1/AE3, rabbit anti-human Vimentin is co-staining with DAPI nucleus;
2) lightly wiping tissue block blood with gauze in a super clean bench, washing with PBS +10% antibiotics, putting the cleaned tissue block into a 6cm culture dish added with 0.5mL of DMEM-high sugar culture solution, and cutting the tissue in the DMEM-high sugar culture solution into fragments of 1mm multiplied by 1 mm;
3) adding 2mL of EDTA-containing 0.05% trypsin covering tissue into the fragments in the step 2), placing the fragments in an incubator with 5% CO2 and 37 ℃, and performing digestion treatment for 25min to obtain a digestion solution containing a cell suspension;
4) filtering the digestive juice containing the cell suspension in the step 3) to a 50mL test tube filled with 2mL fetal calf serum by using a 100um sterile cell filter, washing the digestive juice with PBS once to obtain a digestive filtrate containing the fetal calf serum, and placing the digestive filtrate on ice to enable cells to be in a low-temperature environment;
5) reversely buckling the 100um sterile cell filter in the step 4) on an original culture dish, washing the filter screen of the 100um sterile cell filter by using 0.2% I-type collagenase, completely washing viscous tissues on the filter screen into the culture dish, using 6mL of 0.2% I-type collagenase totally, transferring the washing liquid to a 15mL centrifuge tube, and further digesting the undigested tissues in the centrifuge tube;
6) horizontally placing the centrifugal tube in the step 5) in a shaking box with the temperature of 37 ℃ and the rotating speed of 180r/min to shake for 45min, so that the residual tissue is sufficiently digested, and obtaining tissue fragment digestive juice;
7) adding the digestion filtrate containing fetal calf serum in the step 4) into the tissue fragment digestion solution in the step 6), centrifuging, and removing supernatant to obtain cell sediment;
8) fully blowing and beating the resuspended cell sediment by DMEM high-sugar culture solution, 5% FBS, 1% antibiotic and HEPES 10mM/mL culture solution, counting by a cell counting plate, transferring the obtained cells to a place where 5% CO is placed2Culturing in an incubator at 37 ℃;
9) the separation and passage, the culture observation of the cervical squamous carcinoma tissue cell specimen, and the results are shown in fig. 5, wherein, after 24 hours of culture, the cell attachment of single cell or the cell climbing out of jelly-like tissue can be seen (10X), after 7 days of culture, a 6cm culture dish (4X) can be tiled, after C. digestion and passage, the clone of P1 visible cell grows out (10X), D. P1 (1: 3 passages) and culturing for 3 days, namely spreading a 6cm culture plate with a cobblestone-like appearance (40X);
10) after the cells in the culture dish are digested, magnetic bead sorting is carried out, and identification after culture is carried out, the results are shown in FIG. 6, and the EpCAM/CD326+ cells are identified as cervical squamous carcinoma cells through mouse anti-human AE1/AE3 monoclonal antibodies, rabbit anti-human Vimentin polyclonal antibodies and DAPI blue counterstaining nuclei.
The expression of AE1+ alpha-SMA, AE1+ P16 and P16+ HPV16/18-E6 in the selected cervical squamous cell carcinoma specimen is checked, and the result is shown in FIG. 7, wherein FIG. 7A shows the expression of AE1+ alpha-SMA in the cervical squamous cell carcinoma specimen, and the specimen is oval (20X); FIG. 7B shows the expression of AE1+ P16 in cytology specimen of cervical squamous carcinoma; FIG. 7C shows the expression of cervical squamous carcinoma tissue cell specimen P16+ HPV 16/18-E6.
The cell purity and the cancer stem cell marker were detected for EpCAM/CD326+ sorted by magnetic beads from cervical squamous carcinoma tissue cell specimens, and the results are shown in fig. 8, where the detection purity is: more than 98.72 percent.
The detection of the cell cancer stem cell marker is performed on EpCAM/CD326+ sorted by a plurality of groups of cervical squamous carcinoma tissue cell specimen magnetic beads, and the statistical result is shown in FIG. 9, wherein the cancer stem cell marker detects CD24+ and CD133+ is highly expressed.
The results of multicolor flow cytometry marker analysis of EpCAM/CD 326-sorted by magnetic beads of cervical squamous carcinoma tissue cell specimens are shown in fig. 10, the content of the relevant fibroblasts is 17%, and EpCAM/CD 326-cells sorted by magnetic beads are mixed population cells and comprise fibroblasts and part of tumor cells.
EpCAM/CD 326-sorted multiple groups of magnetic beads of cervical squamous cell carcinoma tissue specimens are used for detecting the fibroblast markers, and the statistical result is shown in figure 11, wherein CD44 in the cancer-related fibroblast population is highly expressed.
The formation of 14d cell clone in the selected cervical squamous carcinoma tissue cell specimen is observed, and the result is shown in FIG. 12, and the cell clone spots are seen to grow out.
Comparison of the human cervical cancer tissue-isolated cell proliferation time with magnetic bead sorting EpCAM/CD326+, EpCAM/CD 326-and unsorted cells showed that PDT required for CD326+ tumor cells was short, as shown in FIG. 13.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (4)
1. A separation and culture method of primary cervical carcinoma tumor cells is characterized by comprising the following steps:
1) placing the cut tissue blocks into a precooling culture solution, and placing at a low temperature;
2) cleaning tissues in a super clean bench, placing the cleaned tissue blocks into a culture dish with culture solution, cutting the tissues into fragments of 1mm multiplied by 1mm to obtain fragment tissues, and washing the tissues by PBS and 10% antibiotics;
3) adding a digestive liquid into the fragment tissues in the step 2), placing the mixture in an incubator for digestion treatment to obtain a digestive liquid containing cell suspension, wherein the digestive liquid is 0.05% trypsin containing EDTA, and the digestion time is 15-30 min;
4) filtering the digestive juice containing the cell suspension in the step 3) into a test tube filled with fetal calf serum by using a 100um sterile cell filter, washing to obtain digestive filtrate containing the fetal calf serum, and placing at a low temperature;
5) reversely buckling the 100um sterile cell filter in the step 4) on a culture dish, washing the filter screen of the 100um sterile cell filter by using 0.2% I-type collagenase, and transferring the washing liquid to a centrifuge tube;
6) horizontally placing the centrifugal tube in the step 5) in a shaking box for fully shaking to obtain a tissue fragment digestive juice, wherein the temperature of the shaking box is 37 ℃, the rotating speed is 180r/min, and the shaking time is 45-60 min;
7) adding the digestion filtrate containing fetal calf serum in the step 4) into the tissue fragment digestion solution in the step 6), centrifuging, and removing supernatant to obtain cell sediment;
8) fully blowing and beating the resuspended cell sediment by using culture solution, and transferring the obtained cells into an incubator for culture;
9) separating and passaging;
10) and (4) magnetic bead sorting, wherein the magnetic bead sorting is EpCAM/CD326 positive tumor cells.
2. The method for separating and culturing primary cells of cervical cancer tumor according to claim 1, wherein the culture solution in step 1), step 2) and step 8) is one of DMEM high-sugar culture solution +5% FBS +1% antibiotic + HEPES 10mM/mL, DMEM/F12 +10% FBS +1% antibiotic or DMEM-high-sugar culture solution.
3. The method for separating and culturing the primary cervical cancer tumor cells according to claim 1, wherein the low temperature storage in step 1) is a refrigerator or an ice bag at 4 ℃.
4. The method for separating and culturing primary cells of cervical cancer tumor according to claim 1, wherein the culture condition of the incubator in step 3) and step 8) is 5% CO2、37℃。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202705370U (en) * | 2011-12-28 | 2013-01-30 | 浙江大学 | Human primary cancer cell separation and culture kit |
| CN103602630A (en) * | 2013-12-04 | 2014-02-26 | 刘玉珍 | In vitro culture method of cervical intraepithelial neoplasia cells |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
| CN105200114A (en) * | 2015-10-26 | 2015-12-30 | 南通市肿瘤医院 | Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell |
| CN106497874A (en) * | 2016-09-28 | 2017-03-15 | 深圳中健生物技术有限公司 | A kind of preparation method of Breast Cancer-Specific CTLs |
| CN106999585A (en) * | 2014-09-28 | 2017-08-01 | 加利福尼亚大学董事会 | Regulation to excitant and non-irritating bone marrow cell |
-
2019
- 2019-01-10 CN CN201910021776.4A patent/CN109609463B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202705370U (en) * | 2011-12-28 | 2013-01-30 | 浙江大学 | Human primary cancer cell separation and culture kit |
| CN103602630A (en) * | 2013-12-04 | 2014-02-26 | 刘玉珍 | In vitro culture method of cervical intraepithelial neoplasia cells |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
| CN106999585A (en) * | 2014-09-28 | 2017-08-01 | 加利福尼亚大学董事会 | Regulation to excitant and non-irritating bone marrow cell |
| CN105200114A (en) * | 2015-10-26 | 2015-12-30 | 南通市肿瘤医院 | Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell |
| CN106497874A (en) * | 2016-09-28 | 2017-03-15 | 深圳中健生物技术有限公司 | A kind of preparation method of Breast Cancer-Specific CTLs |
Non-Patent Citations (2)
| Title |
|---|
| 人***细胞的体外培养及鉴定;马秀萍等;《中国组织工程研究》;20120311;第16卷(第11期);第1960页左栏第1段 * |
| 癌旁组织标志物用于早期诊断***癌的初步研究;刘通;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20180315(第03期);第1.1.4.4.2节 * |
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