CN102604895A - Human primary tumor cell separation and culture kit - Google Patents
Human primary tumor cell separation and culture kit Download PDFInfo
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- CN102604895A CN102604895A CN2011104474291A CN201110447429A CN102604895A CN 102604895 A CN102604895 A CN 102604895A CN 2011104474291 A CN2011104474291 A CN 2011104474291A CN 201110447429 A CN201110447429 A CN 201110447429A CN 102604895 A CN102604895 A CN 102604895A
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Abstract
The invention provides a human primary tumor cell separation and culture kit, consisting of a packing box, a cell culture plate with 6 pores, cleaning solution, trypsinization solution, collagenase-hyaluronidase digestive juice, a cell separating medium, selective tumor culture solution and an operating instruction, wherein sterile RPMI (Roswell Park Memorial Institute) 1640 cell culture solution containing 400U/mL of penicillin and 400 mu g/mL of streptomycin serves as the cleaning solution. The human primary tumor cell separation and culture kit solves the problems that the conventional primary tumor cell culture process is complicated, the purity of obtained tumor cells are not high, and the tumor cells are easy to be contaminated by cells. According to the invention, the design is reasonable, the opportunity of germ contamination is less in the process of separation and culture of primary tumor cells in vitro, and only a few of centrifugal tubes and other common consumable items are needed, so that the kit is simple and convenient to use, the purity of the cultured tumor cells is higher, and the effect is good.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of people's primary tumor cell and separate and the culture reagent box.
Background technology
Tumor cell culture is the important means of research canceration mechanism and cellular biology of tumor characteristic; Application vitro culture technology is carried out tumor research and had many advantages: the influence of internal body factor can be avoided in (1), thereby is convenient to explore the influence of various factorss such as physics, chemistry and biology to the tumour cell vital movement; (2) be convenient to study the 26S Proteasome Structure and Function of tumour cell; (3) but prolonged preservation so that observe the change of tumour cell genetic behavior; (4) can be used for the rapid screening cancer therapy drug.
The tumor cell culture technology is divided into the primary tumor cell cultivation and tumor cell line is cultivated.Primary tumor cell is cultivated and is referred to directly from the vivo tumor sample, cultivate in external environment behind the separating tumor cell; Tumor cell line is cultivated the tumour cell vitro culture that refers to vivo tumor sample source; Continuous purifying; Form the cell strain of biological character and hereditary basically identical; Along with tumour constantly goes down to posterity; The biological property of its cell strain is compared with primary tumor cell and possibly also certain variation can be taken place with inherited character, thus primary tumor cell to cultivate be one of gordian technique of many oncology studies (like the tumour antigen inductive tumor biotherapy of individual tumors drug sensitive test, individuation, the experiment of tumor-specific cytotoxicity T lymphocyte etc.).
Recent study confirms, BMDC (DC) can induce tumor-specific cytotoxicity T lymphocyte (CTL) after by various forms of tumour antigen sensitization in vivo and in vitro, utilizes DC to carry out immunotherapy of tumors at present and has got into clinical experimental stage.GM-CSF and IL-4 combined utilization can induce monocyte to become the DC with prematurity phenotype, give certain stimulus signal effect again, like cytokines such as TNF-α, LPS, can induce ripe DC, make its antigen presentation function reach optimum regime.In the tumor-killing test of cell in vitro cultivation and nude mice animal model for tumour; The DC-CIK of tumour antigen load (BMDC-cytokine induced kill cell) therapy shows remarkable advantages at aspects such as t cell activation, inducing apoptosis of tumour cell than other biotherapy mode such as LAK (lymphokine activated killer cell) treatment etc.; But in clinical application research, also find; The DC-CIK therapy does not obtain the result of treatment as in vitro culture and the animal model; Trace it to its cause; Maybe be relevant with the low MHC I quasi-molecule of expressing of DC, and the MHC I quasi-molecule necessary molecule of submission tumour antigen just also possibly separate with culture technique relevant with present primary tumor cell.In the noumenal tumour tissue except that containing tumour cell; Also comprise cellular constituents such as a large amount of red corpuscle, monocyte, lymphocyte, endotheliocyte, inoblast, tumor tissues is after the digestion of Digestive systems such as collagenase separates, and above-mentioned cell is separated with tumour cell; After the mixed culture; Even through the selective medium screening of tumour cell, the tumour cell purity that is obtained still possibly influence the ability that tumour-specific activates CTL also about 60%～70%.In primary tumor cell separation and culturing process; Bacterial contamination also is serious problems that influence culture success ratio; Tumor specimen comes from the cardinal principle sample of excision more, all might receive cell contamination, especially gastroenteric tumor shearing tumor specimen and be transferred in the breadboard process; Sample itself promptly possibly have a large amount of gastrointestinal tract cells, so needs the extremely problem of cell prevention cell contamination in the primary tumor cell culturing process.
What current primary tumor cell cultural method still adopted is traditional primary cell culture method, and main process comprises: (1) is cleaned sample and tumor specimen is shredded written treaty 1 mm
3Size; (2) with the tumor specimen that shreds with pancreatin or collagenase digesting; (3) collect the cell digest, in the tumour cell special culture media, cultivate behind the purifying in addition with the whole bag of tricks such as density gradient centrifugations.In said process, need use multiple consumptive material and reagent, comprise Tissue Culture Dish or culture plate, digestive ferment, scavenging solution, parting liquid, the grand nutrient solution of selection or the like, be easy to occur the problem of cell contamination.Therefore be necessary the former foster test kit of being commissioned to train of various tumours of developing special.
Summary of the invention
The present invention provides a kind of people's primary tumor cell to separate and the culture reagent box, is made up of packing box, 6 porocyte culture plates, scavenging solution, trysinization liquid, collagenase-Unidasa Digestive system, cellular segregation liquid, tumor-selective nutrient solution, working instructions.
Wherein scavenging solution is aseptic RPMI 1640 cell culture fluids that contain penicillium mould 400U/mL, Streptomycin sulphate 400ug/mL, divides 2 the 5ml sterile seal bottles of packing into, and is labelled, 4 ℃ of preservations.
Trysinization liquid is to contain 0.1% trypsin available from Sigma company with the preparation of aseptic ultrapure water, 100mg), the trysinization liquid of 10g/L Vinylpyrrolidone polymer, divides the 5ml sterile seal bottle of packing into, and is labelled, 4 ℃ of preservations.
Collagenase-Unidasa Digestive system is with RPMI 1640 cell culture fluids preparation collagenase-Unidasa Digestive system; Wherein (available from Sigma company, 100mg) final concentration is 2g/L to collagenase, and Unidasa is (available from Sigma company; 100mg) final concentration is 2g/L; Divide the 5ml sterile seal bottle of packing into, labelled, 4 ℃ of preservations.
Cellular segregation liquid is (available from Pharmacia with pure percoll parting liquid; Be to be that preparation density is 1.09 cellular segregation liquid behind the 7:3 ratio mixing with the volume ratio with 1 times of PBS liquid again after the 9:1 mixed with the volume ratio 100ml) with 10 times of PBS damping fluids; Divide the aseptic brown air-tight bottle of 5ml of packing into; Labelled, 4 ℃ keep in Dark Place.
The tumor-selective nutrient solution is that interpolation 10% newborn foetal calf serum, penicillium mould 100U/mL, Streptomycin sulphate 100ug/mL, Regular Insulin 5ug/mL, HYDROCORTISONE INJECTIONS 2ug/mL, people recombinate Urogastron 5ng/mL (available from Invitrogen company on RPMI 1640 cell culture fluid bases; 100ug); Divide the 5ml sterile seal bottle of packing into; Labelled, 4 ℃ of preservations.
Beneficial effect of the present invention: use the present invention can implement the separation and the cultivation of external primary tumor cell more easily, the tumour cell purity of cultivation is higher, and the chance that receives cell contamination still less.Main technical principle of the present invention is to adopt trysinization liquid and collagenase, the digestion of Unidasa Digestive system successive to improve the digestion effect; 70% percoll parting liquid is removed red corpuscle; Mouse tail collagen and mouse-anti-human T HY-1 antibody hybrid packet are selected culture hole by tumour; With tumor-selective substratum amplification cultivation tumour cell; Thereby obtain the primary tumor cell of based on very high purity; Receive the chance of bacterial contamination few in separation and the culturing process, only need other consumptive materials commonly used such as a small amount of centrifuge tube, therefore have simple and convenient and the good advantage of culture effect.In order to overcome the problem that current primary tumor cell culturing process is complicated, gained tumour purity is not high, be subject to cell contamination, simple and convenient.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Fig. 2 is the present invention's 6 porocyte culture plates numbering synoptic diagram.
Fig. 3 is lymphocyte and monocyte figure adherent in the e hole.
Fig. 4 is tumour cell figure adherent in the f hole.
Embodiment
The present invention combines accompanying drawing and embodiment to be further described.
Embodiment one
Referring to Fig. 1, Fig. 2, test kit is made up of 1,16 porocyte culture plate 2 of packing box, 4,1 bottle of 5ml collagenase of 3,1 bottle of 5ml trysinization liquid of 5ml scavenging solution-5,1 bottle of 5ml cellular segregation liquid of Unidasa Digestive system 6,1 bottle of 5ml tumor-selective nutrient solution 7, working instructions 8.
Wherein scavenging solution 3 is aseptic RPMI 1640 cell culture fluids that contain penicillium mould 400U/mL, contain Streptomycin sulphate 400ug/mL, and 2 the 5ml sterile seal bottles of packing into are labelled, 4 ℃ of preservations.
Collagenase-Unidasa Digestive system 5 is with RPMI 1640 cell culture fluids preparation collagenase-Unidasa Digestive system; Wherein (available from Sigma company, 100mg) final concentration is 2g/L to collagenase, and Unidasa is (available from Sigma company; 100mg) final concentration is 2g/L; Divide the 5ml sterile seal bottle of packing into, labelled, 4 ℃ of preservations.
Cellular segregation liquid 6 is (available from Pharmacia with pure percoll parting liquid; Be to be that preparation density is 1.09 cellular segregation liquid behind the 7:3 ratio mixing with the volume ratio with 1 times of PBS liquid again after the 9:1 mixed with the volume ratio 100ml) with 10 times of PBS damping fluids; Divide the aseptic brown air-tight bottle of 5ml of packing into; Labelled, 4 ℃ keep in Dark Place.
Tumor-selective nutrient solution 7 is that interpolation 10% newborn foetal calf serum, penicillium mould 100U/mL, Streptomycin sulphate 100ug/mL, Regular Insulin 5ug/mL, HYDROCORTISONE INJECTIONS 2ug/mL, people recombinate Urogastron 5ng/mL (available from Invitrogen company on RPMI 1640 cell culture fluid bases; 100ug); Divide the 5ml sterile seal bottle of packing into; Labelled, 4 ℃ of preservations.
6 porocyte culture plates 2 can purchase the common 6 porocyte culture plates in market in addition sterilization or import sterilized independent packaging 6 porocyte culture plates (like U.S. CORNING, article No.: 3516).Do not add any processing for earlier the hole wall in a, b, c, d, f hole in 6 porocyte culture plates, 2 every hole numbering (a-f) 6 porocyte culture plates 2 before using this test kit; The e hole encapsulates hole wall with mouse tail collagen protein mixing mouse-anti-human T HY-1 monoclonal antibody; Method for coating: (give birth to friendly Bioisystech Co., Ltd available from Hangzhou with aseptic 0.006mol/L (0.36g/L) acetate preparation mouse tail collagen and anti-THY-1 monoclonal antibody mixed solution mouse tail collagen protein; 10mg) final concentration is that (available from the auspicious neat bio tech ltd in Shanghai, 1mg) final concentration is 5ug/ml for 12ug/ml and the anti-mouse THY-1 of people antibody; Add 1.5ml tail collagen protein and anti-THY-1 monoclonal antibody mixed solution in the e hole, uncapping spends the night on super clean bench dries, and dries after washing 3-4 time with PBS, and 1 the property packing bag for medical use (available from Shenzhen security packing Co., Ltd.) of packing into seals.
Embodiment two
Referring to Fig. 2, use this test kit: give 6 porocyte culture plates 2 every hole numberings (a-f) earlier.
(1) cut tumor specimen after, put into the wherein a hole of 6 porocyte culture plates 2, pour scavenging solution 3 into and cover samples;
(2) in the 1st hole, clean tumor specimen, sample is moved into the b hole, pour scavenging solution 3 into, and tumor specimen is shredded or be cut into 1mm
3Size;
(3) the tumor specimen piece that cleaning is shredded moves into the c hole, pours trysinization liquid 4 into, and predigestion is 15 minutes in 37 ℃ of cell culture incubators;
(4) will pass through predigested tumor specimen behind the emigrated cells incubator and move into the d hole, pour collagenase-Unidasa Digestive system 5 into, and in 4 ℃ of refrigerators, digest and spend the night;
(5) after the digested overnight, collect the cell suspension that digests, warp 200 order cell strainer filterings are to the 50ml centrifuge tube; In the 15ml centrifuge tube, add cellular segregation liquid 6 earlier, slowly add filterable cell suspension on the upper strata of cellular segregation liquid 6, last whizzer separates (2000 rev/mins; 15 minutes), what sink to the centrifuge tube bottom is red corpuscle, a visible muddy cellular layer between cellular segregation liquid and Digestive system; Be the cell mixing layer that comprises tumour cell, draw the cell suspension of cell mixing layer, clean 2 times and remove Digestive system and parting liquid; Add tumor-selective nutrient solution 7, cell counting count board counting, adjustment cell density to 1 * 10
6/ L;
(6) mixed cell suspension is moved into the e hole, put into 37 ℃, 5% CO
2Cell culture incubator in 2 hours;
Monocyte, lymphocyte, endotheliocyte, inoblast will be attached on the hole wall after (7) 2 hours, and the tumour cell suspension growth moves into the f hole behind the tumour cell that absorption suspends, put into 37 ℃, 5% CO
2Cell culture incubator in continue to cultivate, finally obtain highly purified well-grown tumour cell.The result is referring to Fig. 3, among the figure: A is monocyte (greatly), and B is lymphocyte (little).
Embodiment three
Get the liver cancer tissue sample, operation steps finally obtains the liver cancer cell that high purity is the suspension growth with embodiment 2.The result is referring to Fig. 4, is former generation liver cancer cell of cultivating in the f hole, among the figure: the liver cancer cell of C for suspending.
Claims (2)
1. people's primary tumor cell separates and the culture reagent box, is made up of packing box (1), 6 porocyte culture plates (2), scavenging solution (3), trysinization liquid (4), collagenase-Unidasa Digestive system (5), cellular segregation liquid (6), tumor-selective nutrient solution (7), working instructions (8); Wherein said scavenging solution (3) is aseptic RPMI 1640 cell culture fluids that contain penicillium mould 400U/mL, Streptomycin sulphate 400ug/mL; Trysinization liquid (4) is the trysinization liquid that contains 0.1% trypsinase, 10g/L Vinylpyrrolidone polymer with aseptic ultrapure water preparation; Collagenase-Unidasa Digestive system (5) is that the collagenase final concentration is 2g/L with the preparation of RPMI 1640 cell culture fluids, and the Unidasa final concentration is 2g/L; Cellular segregation liquid (6) is to be to be that preparation density is 1.09 cellular segregation liquid behind the 7:3 ratio mixing with the volume ratio with 1 times of PBS liquid again after the 9:1 mixed with the volume ratio with pure percoll parting liquid and 10 times of PBS damping fluids; Tumor-selective nutrient solution (7) is in RPMI 1640 cell culture fluids, to add 10% newborn foetal calf serum, penicillium mould 100U/mL, Streptomycin sulphate 100ug/mL, Regular Insulin 5ug/mL, HYDROCORTISONE INJECTIONS 2ug/mL, the people Urogastron 5ng/mL that recombinates.
2. a kind of people's primary tumor cell according to claim 1 separates and the culture reagent box; It is characterized in that; Saidly contain penicillium mould scavenging solution (3), contain Streptomycin sulphate scavenging solution (4), trysinization liquid (5), collagenase-Unidasa Digestive system (6), cellular segregation liquid (7), tumor-selective nutrient solution (8); Divide equally the 5ml sterile seal bottle of packing into, labelled, 4 ℃ of preservations.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468746A (en) * | 2013-09-28 | 2013-12-25 | 青岛麦迪赛斯生物科技有限公司 | Method for constructing tumor cell line |
| CN104111254A (en) * | 2014-07-09 | 2014-10-22 | 李维 | Tumor chemotherapy drug susceptibility detection kit |
| CN108070560A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary stomach cancer cell of people |
| CN111808816A (en) * | 2019-04-11 | 2020-10-23 | 北京基石生命科技有限公司 | A medium for culturing gastric cancer solid tumor primary cells |
| RU2782476C2 (en) * | 2016-01-18 | 2022-10-28 | Аморфикал Лтд. | Stabilized amorphous calcium carbonate for use as additive for cellular cultural media |
| US12060573B2 (en) | 2016-01-18 | 2024-08-13 | Amorphical Ltd. | Stabilized amorphous calcium carbonate as a supplement for cell culture media |
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2011
- 2011-12-28 CN CN 201110447429 patent/CN102604895B/en not_active Expired - Fee Related
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| 刘浩等: "人非小细胞肺癌细胞的原代培养及个体化药敏实验研究", 《中华肺部疾病杂志》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468746A (en) * | 2013-09-28 | 2013-12-25 | 青岛麦迪赛斯生物科技有限公司 | Method for constructing tumor cell line |
| CN104111254A (en) * | 2014-07-09 | 2014-10-22 | 李维 | Tumor chemotherapy drug susceptibility detection kit |
| RU2782476C2 (en) * | 2016-01-18 | 2022-10-28 | Аморфикал Лтд. | Stabilized amorphous calcium carbonate for use as additive for cellular cultural media |
| US12060573B2 (en) | 2016-01-18 | 2024-08-13 | Amorphical Ltd. | Stabilized amorphous calcium carbonate as a supplement for cell culture media |
| CN108070560A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary stomach cancer cell of people |
| CN111808816A (en) * | 2019-04-11 | 2020-10-23 | 北京基石生命科技有限公司 | A medium for culturing gastric cancer solid tumor primary cells |
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| CN102604895B (en) | 2013-07-31 |
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