CN102796704B - Temperature control cell culture method - Google Patents
Temperature control cell culture method Download PDFInfo
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- CN102796704B CN102796704B CN201210320799.3A CN201210320799A CN102796704B CN 102796704 B CN102796704 B CN 102796704B CN 201210320799 A CN201210320799 A CN 201210320799A CN 102796704 B CN102796704 B CN 102796704B
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Abstract
The invention discloses a temperature control cell culture method, which belongs to the technical field of cell biology. The temperature control cell culture method comprises the following process steps of: preparing a micro-carrier; inoculating cells; performing temperature control of cell culture; and detecting a cell proliferation condition. The temperature control cell culture method has the advantages that the cells are kept in a sleep state through temperature control under some special conditions, for example, during spatial cell carrying or before aircraft injection, are not affected by an external environment and are activated through temperature control after the aircraft injection, so that more living cells can be obtained and sufficient experimental material is guaranteed to perform subsequent experiment, the cells are only affected by the spatial environment, the influence on the cells before aircraft injection is reduced, the influence on the cells by space environment can be more accurately responded, meanwhile, extra operation of astronauts is not required, and the workload and the working difficulty of the astronauts are reduced.
Description
Technical field
The invention belongs to cytobiology technology field, a kind of temperature control cell culture processes is particularly provided, be applicable to space and carry cell cultures.
Technical background
Along with the development of China Aerospace cause, the research of space biology benefit is more and more deep, utilize at present retrievable satellite and Shenzhou spacecraft lift-launch plant seed and microorganism to carry out large quantity research, but for humans and animals cell, because culture condition is limit, research less, particularly rarely has report especially through the research that satellite and Spaceship Carrying obtain viable cell.
Because aircraft is comparatively complicated in transmitting and the precondition of entering the orbit, can make cell be affected, researchist often wishes at this phase cell, in dormant state, after aircraft is entered the orbit, cell to be activated again.At present domestic also have some cells to carry culture apparatus, but all need spacefarer's manual operation, needs spacefarer's timing to add activator to guarantee the growth of phase cell in-orbit.
Summary of the invention
Object of the present invention is to provide a kind of temperature control cell culture processes, carries out cell cultures by temperature control, and processing step is as follows:
1. microcarrier is prepared
Take 100-500 milligram microcarrier (being made by one or more in Mierocrystalline cellulose, mucopolysaccharide, collagen, polystyrene, silicon, acrylamide) in centrifuge tube, add 10-40 ml phosphate buffer (DPBS, made by the sodium-chlor of 1-8 grams per liter, the Repone K of 0.1-0.2 grams per liter, the Sodium phosphate dibasic of 1-2 grams per liter, the potassium primary phosphate of 0.1-0.2 grams per liter), after soaking at room temperature 1-4 hour, the phosphate buffered saline buffer more renewing cleans after 2 times, remove phosphate buffered saline buffer, put into high-pressure sterilizing pot 115-121 ℃ of sterilizing 15-30 minute.After taking out, remove excessive moisture, add the perfect medium balance 10-24h containing percent by volume 5-10% foetal calf serum, obtaining can be for the microcarrier of Growth of Cells;
2. cell inoculation
Get tumour cell in logarithmic phase as mouse melanin tumor cell (B16 cell), renal epithelial cell (293 cell), human lung carcinoma cell (H1299) etc., or primary cultured cell is as human skin fibroblast (ESF cell), mouse embryo fibroblasts (3T3-L1 cell), cell quantity is 1-10 × 10
6individual, use 0.05-0.25%(percent by volume) tryptic digestion after, by cell counting count board counting cells quantity, use containing 1-15%(percent by volume) to adjust cell quantity be 0.5-2 × 10 for the perfect medium of foetal calf serum
5individual cells/ml, the microcarrier that can be used for Growth of Cells that adds step 1 to obtain, making its concentration is 1-10 mg/ml, obtains the mixture of cell and microcarrier.This mixture is placed in culture dish, puts into 37 ℃, 5%CO
2incubator in, every 15-30 minute rocks culture dish No. one time in 1-5 hour, and cell is contacted completely with microcarrier.Cultivate after 4-16 hour, cell is all attached on microcarrier and grows;
3. the temperature control of cell cultures
What step 2 was obtained grows in the cell on microcarrier, proceed in new culture dish, add containing 1% or 10%(percent by volume) perfect medium of foetal calf serum, cover lid, sealed membrane sealing, is positioned over respectively in 20-25 ℃ and 37 ℃ of temperature regulators.
4. cell proliferation situation detects
Grow in the cell on microcarrier, in 20-25 ℃ and 37 ℃ of temperature regulators, place after 24-240 hour, taking-up shakes up, get 100-1000 microlitre cell suspension, add 1-15%(percent by volume) CCK-8, hatch 1-4 hour for 25-37 ℃, get supernatant liquor in enzyme plate, use microplate reader detects the OD value of supernatant liquor, the propagation of OD value reacting cells under 450-550 nano wave length.Now cell is not bred, and cell is in dormant state, but still has activity.
To change the perfect medium containing 10% foetal calf serum into containing the substratum of 1% foetal calf serum, temperature regulator is warming up to 37 ℃ and continues to cultivate 24-240 hour, get the above-mentioned CCK-8 method of 100-1000 microlitre cell suspension and detect cell proliferation situation, now cell has started propagation, and there is no significant difference with the cellular control unit that is positioned over 37 ℃ of cultivations always.
Microcarrier of the present invention is spherical, and diameter is at 60-87 micron.
Described CCK-8 of the present invention is a kind of for measuring cell proliferation or the highly sensitive colorimetric detection method of toxicity test viable count object, and its colour-change is directly proportional to cell quantity.
The invention has the advantages that, under some special conditionss, while lift-launch as spatial cell, before aircraft is entered the orbit, by temperature control, make cell in dormant state, cell is not affected by the external environment, aircraft after entering the orbit again by temperature control activating cells, can not only obtain so more viable cell, guarantee to have enough experiment materials to carry out subsequent experimental, cell is only subject to the impact of space environment simultaneously, reduce the impact that cell is subject to before airship is entered the orbit, more can the impact of accurate response space environment on cell, do not need the extra operation of spacefarer simultaneously, less spacefarer's workload and work difficulty.
Accompanying drawing explanation
Fig. 1 is the propagation of B16 cell use temperature control method culturing cell.
Fig. 2 is the propagation of ESF cell use temperature control method culturing cell.
Cultivate after 5 days at 20-23 ℃, B16 cell and ESF cell are not bred substantially, and in dormant state, but cell still has activity.Use the B16 cell of the culture medium culturing of 1% foetal calf serum, also without had significant proliferation, but before 37 ℃, need to use instead the perfect medium containing 10% foetal calf serum being warming up to.Cell 37 ℃ of placements has had significant proliferation.When temperature regulator is warmed up to after 37 ℃, the cell that was originally put in 20-23 ℃ is activated, and starts propagation.
Embodiment
Embodiment:
1. microcarrier is prepared
Take 500 milligrams of microcarriers in centrifuge tube, add 40 ml phosphate buffers, soaking at room temperature is after 2 hours, and the phosphate buffered saline buffer more renewing cleans after 2 times, removes phosphate buffered saline buffer, puts into 120 ℃ of sterilizings of high-pressure sterilizing pot 20 minutes.After taking out, remove excessive moisture, add the perfect medium balance 16h containing 10% foetal calf serum, obtaining can be for the microcarrier of Growth of Cells;
2. cell inoculation
Get mouse melanin tumor cell (B16 cell) and primary cultivator skin flbroblast (ESF cell) in logarithmic phase, with after 0.25% tryptic digestion, by cell counting count board counting cells quantity, using and adjusting cell quantity containing the perfect medium of 10% foetal calf serum is 1 × 10
5individual cells/ml, adds the microcarrier that can be used for Growth of Cells, and making its concentration is 5 mg/ml, obtains the mixture of cell and microcarrier.This mixture is placed in culture dish, puts into 37 ℃, 5%CO
2incubator in, within every 20 minutes, rock culture dish No. one time in 3 hours, cell is fully contacted with microcarrier.Cultivate after 16 hours, cell is all attached on microcarrier and grows;
3. the temperature control of cell cultures
By the cell growing on microcarrier, proceed in new culture dish, add the perfect medium containing 1% or 10% foetal calf serum, cover lid, sealed membrane sealing, is positioned over respectively in 20-23 ℃ and 37 ℃ of temperature regulators.
4. cell proliferation situation detects
Grow in the cell on microcarrier, in 20-23 ℃ and 37 ℃ of temperature regulators, place after 120 hours, taking-up shakes up, get 180 microlitre cell suspensions, add 10% CCK-8, hatch 1 hour, get supernatant liquor in enzyme plate for 37 ℃, use microplate reader detects the OD value of supernatant liquor, the propagation of OD value reacting cells under 450 nano wave lengths.Now cell is not bred, and cell is in dormant state, but still has activity.
To change the perfect medium containing 10% foetal calf serum into containing the perfect medium of 1% foetal calf serum, temperature regulator is warming up to 37 ℃ to be continued to cultivate 96 hours, get the above-mentioned CCK-8 method of 180 microlitre cell suspensions and detect cell proliferation situation, now cell has started propagation, and there is no significant difference with the cellular control unit that is positioned over 37 ℃ of cultivations always.
Claims (7)
1. a temperature control cell culture processes, is characterized in that, processing step is as follows:
(1) microcarrier is prepared
Take 100-500 milligram microcarrier in centrifuge tube, add 10-40 ml phosphate buffer DPBS, after soaking at room temperature 1-4 hour, the phosphate buffered saline buffer DPBS more renewing cleans after 2 times, remove phosphate buffered saline buffer DPBS, put into high-pressure sterilizing pot 115-121 ℃ of sterilizing 15-30 minute; After taking out, remove excessive moisture, add the perfect medium balance 10-24h containing percent by volume 5-10% foetal calf serum, obtain the microcarrier for Growth of Cells;
(2) cell inoculation
Get tumour cell or primary cultured cell in logarithmic phase, cell quantity is 1-10 × 10
6individual, with after the tryptic digestion of 0.05-0.25%, by cell counting count board counting cells quantity, using and adjusting cell quantity containing the perfect medium of 1-15% foetal calf serum is 0.5-2 × 10
5individual cells/ml, the microcarrier for Growth of Cells that adds step (1) to obtain, making its concentration is 1-10 mg/ml, obtains the mixture of cell and microcarrier; This mixture is placed in culture dish, puts into 37 ℃, 5%CO
2incubator in, every 15-30 minute rocks culture dish No. one time in 1-5 hour, and cell is contacted completely with microcarrier; Cultivate after 4-16 hour, cell is all attached on microcarrier and grows;
(3) the temperature control of cell cultures
What step (2) was obtained grows in the cell on microcarrier; proceed in new culture dish; add the perfect medium containing 1% or 10% foetal calf serum; cover lid; sealed membrane sealing; the cell that contains the perfect medium of 1% foetal calf serum is positioned in 20-25 ℃ of temperature regulator, and the cell that contains the perfect medium of 10% foetal calf serum is positioned in 37 ℃ of temperature regulators;
(4) cell proliferation situation detects
Grow in the cell on microcarrier, in 20-25 ℃ and 37 ℃ of temperature regulators, place after 24-240 hour, taking-up shakes up, get 100-1000 microlitre cell suspension, add the CCK-8 of 1-15%, hatch 1-4 hour for 25-37 ℃, get supernatant liquor in enzyme plate, use microplate reader detects the OD value of supernatant liquor, the propagation of OD value reacting cells under 450-550 nano wave length; Now, the cell of initial incubation temperature under 20-25 ℃ of condition do not bred, and cell is in dormant state, but still has activity;
To change the perfect medium containing 10% foetal calf serum into containing the substratum of 1% foetal calf serum, temperature regulator is warming up to 37 ℃ and continues to cultivate 24-240 hour, cultured cells in 37 ℃ of temperature regulators always, continues to cultivate 24-240 hour in 37 ℃ of temperature regulators; Taking-up shakes up, get the above-mentioned CCK-8 method of 100-1000 microlitre cell suspension and detect cell proliferation situation, now, the cell of initial incubation temperature under 20-25 ℃ of condition started propagation, and in cell proliferation situation and form, there is no significant difference with the cellular control unit that is positioned over 37 ℃ of cultivations always.
2. temperature control cell culture processes according to claim 1, is characterized in that, described microcarrier is spherical, and diameter is at 60-87 micron.
3. temperature control cell culture processes according to claim 1, is characterized in that, described CCK-8 is a kind of for measuring cell proliferation or the highly sensitive colorimetric detection method of toxicity test viable count object, and its colour-change is directly proportional to cell quantity.
4. temperature control cell culture processes according to claim 1, is characterized in that, described microcarrier is made up of one or more in Mierocrystalline cellulose, mucopolysaccharide, collagen, polystyrene, silicon, acrylamide.
5. temperature control cell culture processes according to claim 1, it is characterized in that, described phosphate buffered saline buffer DPBS is made up of the sodium-chlor of 1-8 grams per liter, the Repone K of 0.1-0.2 grams per liter, the Sodium phosphate dibasic of 1-2 grams per liter, the potassium primary phosphate of 0.1-0.2 grams per liter.
6. temperature control cell culture processes according to claim 1, is characterized in that, described tumour cell is B16 mouse melanoma cell line cell, renal epithelial cell 293 cells, human lung carcinoma cell H1299.
7. temperature control cell culture processes according to claim 1, is characterized in that, described primary cultured cell is human skin fibroblast ESF cell, mouse embryo fibroblasts 3T3-L1 cell.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434932A (en) * | 2008-12-22 | 2009-05-20 | 云南省农业科学院甘蔗研究所 | Preparation of sugarcane cell suspending liquid |
| CN101965394A (en) * | 2008-02-21 | 2011-02-02 | Eco解决方案公司 | Method and device for cell culture in the open continuous mode |
| CN102154197A (en) * | 2011-03-02 | 2011-08-17 | 马忠仁 | Baby hamster kidney (BHK)-21 cells obtained by high-density suspension culture in low-serum and serum-free culture medium and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101965394A (en) * | 2008-02-21 | 2011-02-02 | Eco解决方案公司 | Method and device for cell culture in the open continuous mode |
| CN101434932A (en) * | 2008-12-22 | 2009-05-20 | 云南省农业科学院甘蔗研究所 | Preparation of sugarcane cell suspending liquid |
| CN102154197A (en) * | 2011-03-02 | 2011-08-17 | 马忠仁 | Baby hamster kidney (BHK)-21 cells obtained by high-density suspension culture in low-serum and serum-free culture medium and preparation method thereof |
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Effective date of registration: 20170914 Address after: 100081, room 14, building 31, 501-517 South Avenue, Haidian District, Beijing, Zhongguancun Patentee after: SPACE SHENZHOU BIOLOGY & TECHNOLOGY GROUP CO.,LTD. Address before: 100900, Beijing Zhichun Road Haidian District No. 61 Hospital Research Building 2 Patentee before: Beijing Tianchen Space Biological Medical Research Development Co.,Ltd. |
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