CN106520678B - Kit for culturing hepatic stem cells - Google Patents
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Abstract
The invention provides a kit for culturing hepatic stem cells, which is internally provided with a plurality of reagent bottles, wherein the reagent bottles are respectively filled with culture solution, culture medium additives, growth factors I, growth factors II, digestive juice, cleaning solution, frozen stock solution and cell seeds. The kit provided by the invention can be used for culturing, expanding and freezing and storing the liver-derived stem cells, solves the problems of low culturing efficiency, large difference between culturing batches, unsatisfactory revival rate after freezing and storing and the like of the liver-derived stem cells, enables the liver-derived stem cells to be efficiently expanded in an ideal nutrition balance state, greatly saves the culturing and expanding time, is very suitable for culturing the liver-derived stem cells, and provides a rich source for further experimental research and medical treatment by utilizing the liver-derived stem cells.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for culturing hepatic stem cells.
Background
The prior art shows that liver stem cells exist in mature livers, and have strong regeneration capacity. The hepatic stem cell is a stem cell with self-renewal capacity and multidirectional differentiation potential, can generate stem cells with completely the same phenotype and genotype as the hepatic stem cell by self-symmetrical and asymmetrical division, can also generate cells in different development stages and different differentiation directions in liver tissues, such as liver cells, bile duct epithelial cells, pancreatic epithelial cells and the like, can replace liver cell transplantation and liver transplantation to become an important cell source for treating organ failure, and has huge potential in the aspects of liver cell transplantation, in-vitro artificial liver and gene therapy. In the prior art, liver stem cells cultured by using liver tissues of aborted fetuses are used as a precedent for treating diabetes, and good curative effect is obtained; the stem cell injection prepared from the liver stem cells can also be clinically used for treating liver cirrhosis, diabetes and the like.
To meet the needs of medical research and treatment, mass culture of hepatic stem cells has become an important research topic. If the reagent and the method selected by the experimenter for culturing and preserving the hepatic stem cells are not appropriate enough, the quantity and the quality of the cultured hepatic stem cells can not meet the requirements, and the manpower and other resources are wasted, so that the research on the kit specially used for stably amplifying and culturing the hepatic stem cells in large quantities has very important significance. At present, kits specially used for culturing hepatic stem cells are few, some kits used for culturing other stem cells are disclosed in the prior art, for example, CN104928238A discloses a kit used for preparing adipose-derived mesenchymal stem cells, which comprises adipose preservation solution, adipose washing solution, adipose decomposition solution, cell washing solution, cell culture solution, cell digestive juice and cell freezing solution, but the kit has the problems of unsatisfactory effect of stem cell culture and amplification, low reagent safety and stability, low cell survival rate and proliferation rate and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for culturing hepatic stem cells, which is internally provided with a plurality of reagent bottles, wherein the reagent bottles are respectively filled with a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds, and the culture medium additive comprises the following components in parts by weight:
the culture medium additive in the reagent kit can effectively improve the proliferation capacity and the proliferation efficiency of a complete culture medium prepared by the culture medium additive.
Preferably, the culture solution is a DMEM/F12 culture medium solution, the DMEM/F12 culture medium solution is prepared by dissolving a DMEM/F12 dry powder culture medium with water for injection, and 45-60g of the DMEM/F12 dry powder culture medium is dissolved in each L of the water for injection.
Preferably, the growth factor I is bFGF (basic fibroblast growth factor) solution, the bFGF solution is formed by dissolving bFGF freeze-dried powder with water for injection, and 0.1-0.2mg of the bFGF freeze-dried powder is dissolved in each L of the water for injection.
Preferably, the growth factor II is EGF (epidermal growth factor) solution, the EGF solution is formed by dissolving EGF freeze-dried powder with water for injection, and each L of the water for injection dissolves 0.1-0.2mg of the EGF freeze-dried powder.
Preferably, the digestive juice is pancreatin.
Preferably, the washing solution is PBS buffer.
The PBS buffer solution is a phosphate buffer solution containing 0.05% Tween-20 and having a pH value of 7.4.
Preferably, the cell seed is a LSC primary cell.
Further, the culture medium additive also comprises the following components in parts by weight:
synephrine 0.3-1
0.1-0.7 of glycine.
Synephrine with molecular formula C9H13NO2CAS number 94-07-5;
the addition of synephrine and glycine to the culture medium additive is beneficial to further improving the proliferation capacity and the proliferation efficiency of the complete culture medium prepared by the culture medium additive.
Further, the culture medium additive also comprises the following components in parts by weight:
hydroxypropyl cyclodextrin 0.3-0.8
Starch sodium glycolate 0.1-0.5.
The hydroxypropyl cyclodextrin and the sodium starch glycolate are added into the culture medium additive, so that the stability of the complete culture medium prepared by the method can be obviously improved, the problems of precipitation, layering, color change and the like of the complete culture medium are avoided, one component is reduced, or one component is changed, and the stability of the complete culture medium is reduced.
Further, the culture medium additive also comprises the following components in parts by weight:
p-aminobenzoic acid 0.2-0.5
0.1-0.2 of sodium citrate.
The addition of the para aminobenzoic acid and the sodium citrate into the culture medium additive can effectively improve the antibacterial capacity of the complete culture medium prepared by adopting the culture medium additive, effectively inhibit the breeding and reproduction of bacteria in the complete culture medium, ensure the use safety of the complete culture medium, and reduce one component or change one component, which can cause the antibacterial capacity of the complete culture medium to be weakened.
Further, the frozen stock solution is mainly prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, chitosan oligosaccharide and benzalkonium chloride with water for injection, wherein each L of the water for injection is used for dissolving 30-50g of DMEM/F12 dry powder culture medium, 3-8g of dimethyl sulfoxide, 3-6g of chitosan oligosaccharide and 0.2-0.5g of benzalkonium chloride.
The frozen stock solution prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, chitosan oligosaccharide and benzalkonium chloride in water for injection has good freezing performance, the survival rate of the liver-derived stem cells frozen by the frozen stock solution after recovery is high, and the maintenance and the stability of the cell morphology before and after the freezing of the liver-derived stem cells are facilitated.
Further, the frozen stock solution also contains methylcellulose and calcium glycerophosphate, wherein 2-5g of the methylcellulose and 0.2-0.8g of the calcium glycerophosphate are dissolved in each L of the water for injection.
The addition of methylcellulose and calcium glycerophosphate to the frozen stock solution can significantly improve the stability of the frozen stock solution, reduce one of the components, or change one of the components, so that the stability of the frozen stock solution is reduced.
Furthermore, the frozen stock solution also contains sodium stearate lactate and potassium sorbate, wherein 0.2-0.4g of sodium stearate lactate and 0.1-0.3g of potassium sorbate are dissolved in each L of the water for injection.
The sodium stearate lactate and the potassium sorbate are added into the frozen stock solution, so that the antibacterial capacity of the frozen stock solution can be improved, the breeding and propagation of bacteria in the frozen stock solution can be effectively inhibited, and the use safety of the frozen stock solution can be guaranteed.
The invention also provides a method for culturing the liver-derived stem cells, which comprises the following steps:
s1, mixing a culture solution, a culture medium additive, a growth factor I and a growth factor II according to a weight ratio of 3: 0.01: 1: 1, preparing a complete culture medium, and storing the complete culture medium in a refrigerator at 4 ℃;
s2, adding the cell seeds into a cleaning solution, centrifuging for 5min at 4 ℃ and 1000rpm, collecting cells, adding the collected cells into a complete culture medium for resuspension, and then performing resuspension according to the proportion of 1-2 multiplied by 106Per mL ofThe density of the seed is inoculated in a culture flask at 37 ℃ and 5% C02In the environment of (2), hatching cells by using a standard incubator, and replacing the complete culture medium every 24 hours in the hatching process;
s3, when the cell growth area reaches 85-95%, cleaning for 2 times by using a cleaning solution, adding 5% of digestive juice, appropriately shaking and shaking the culture bottle to enable the digestive juice to be fully contacted with the cells, then putting the cells into an incubator to be digested for 1-2min, finally adding a complete culture medium preheated to 37 ℃ to stop digestion, and then carrying out passage on the obtained cells according to the proportion of 1: 3;
s4, repeating the step S3 for 1 time, then centrifuging for 5min at 4 ℃ and 1000rpm, repeating the centrifugation for 2 times, then collecting cells, putting the cells into a freezing medium for freezing and storing, wherein the freezing density is 1-2 multiplied by 107One cell/mL of the frozen stock solution.
The kit provided by the invention can be used for culturing, expanding and freezing and storing the liver-derived stem cells, solves the problems of low culturing efficiency, large difference between culturing batches, unsatisfactory revival rate after freezing and storing and the like of the liver-derived stem cells, enables the liver-derived stem cells to be efficiently expanded in an ideal nutrition balance state, greatly saves the culturing and expanding time, is very suitable for culturing the liver-derived stem cells, and provides a rich source for further experimental research and medical treatment by utilizing the liver-derived stem cells.
Detailed Description
Example 1
A kit for culturing liver-derived stem cells is provided with a plurality of reagent bottles, wherein a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the reagent bottles, and the culture medium additive comprises the following components in parts by weight:
example 2
A kit for culturing liver-derived stem cells is provided with a plurality of reagent bottles, wherein a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the reagent bottles, and the culture medium additive comprises the following components in parts by weight:
example 3
A kit for culturing liver-derived stem cells is provided with a plurality of reagent bottles, wherein a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the reagent bottles, and the culture medium additive comprises the following components in parts by weight:
example 4
A kit for culturing liver-derived stem cells, which is different from example 1 in that: the culture medium additive also comprises the following components in parts by weight:
synephrine 0.3g
0.1g of glycine.
Example 5
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
synephrine 0.5g
0.5g of glycine.
Example 6
A kit for culturing liver-derived stem cells, which is different from example 4 in that: the culture medium additive also comprises the following components in parts by weight:
hydroxypropyl Cyclodextrin 0.3g
Starch glycolate 0.1 g.
Example 7
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
hydroxypropyl Cyclodextrin 0.6g
Starch glycolate 0.2 g.
Example 8
A kit for culturing liver-derived stem cells, which is different from example 6 in that: the culture medium additive also comprises the following components in parts by weight:
p-aminobenzoic acid 0.2g
0.1g of sodium citrate.
Example 9
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
p-aminobenzoic acid 0.3g
0.15g of sodium citrate.
Example 10
A kit for culturing liver-derived stem cells, which is different from example 8 in that: the frozen stock solution is mainly prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, chitosan oligosaccharide and benzalkonium chloride in water for injection, wherein each L of the water for injection is used for dissolving 30g of DMEM/F12 dry powder culture medium, 3g of dimethyl sulfoxide, 3g of chitosan oligosaccharide and 0.2g of benzalkonium chloride.
Example 11
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the frozen stock solution is mainly prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, chitosan oligosaccharide and benzalkonium chloride in water for injection, wherein each L of the water for injection is used for dissolving 40g of DMEM/F12 dry powder culture medium, 5g of dimethyl sulfoxide, 5g of chitosan oligosaccharide and 0.3g of benzalkonium chloride.
Example 12
A kit for culturing liver-derived stem cells, which is different from example 10 in that: the frozen stock solution also contains methylcellulose and calcium glycerophosphate, wherein 2g of the methylcellulose and 0.2g of the calcium glycerophosphate are dissolved in each L of the water for injection.
Example 13
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contains methylcellulose and calcium glycerophosphate, wherein 4g of the methylcellulose and 0.5g of the calcium glycerophosphate are dissolved in each L of the water for injection.
Example 14
A kit for culturing liver-derived stem cells, which is different from example 12 in that: the frozen stock solution also contains sodium stearate lactate and potassium sorbate, wherein 0.2g of sodium stearate lactate and 0.1g of potassium sorbate are dissolved in each L of the water for injection.
Example 15
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contains sodium stearate lactate and potassium sorbate, wherein 0.3g of sodium stearate lactate and 0.2g of potassium sorbate are dissolved in each L of the water for injection.
Example 16
A method for culturing liver-derived stem cells, comprising the steps of:
s1, mixing a culture solution, a culture medium additive, a growth factor I and a growth factor II according to a weight ratio of 3: 0.01: 1: 1, preparing a complete culture medium, and storing the complete culture medium in a refrigerator at 4 ℃;
s2, adding the cell seeds into a cleaning solution, centrifuging for 5min at 4 ℃ and 1000rpm, collecting cells, adding the collected cells into a complete culture medium for resuspension, and then resuspending according to the proportion of 1 multiplied by 106The cells/mL were inoculated in culture flasks at 37 ℃ with 5% C02In the environment of (2), hatching cells by using a standard incubator, and replacing the complete culture medium every 24 hours in the hatching process;
s3, when the cell growth area reaches 85%, cleaning for 2 times by using a cleaning solution, adding 5% of digestive juice, appropriately shaking and shaking the culture bottle to enable the digestive juice to be fully contacted with the cells, then placing the cells into an incubator to be digested for 1min, finally adding a complete culture medium preheated to 37 ℃ to stop the digestion, and then carrying out passage on the obtained cells according to the ratio of 1: 3;
s4, repeating the step S3 for 1 time, then centrifuging for 5min at 4 ℃ and 1000rpm, repeating the centrifugation for 2 times, then collecting cells, putting the cells into a freezing medium for freezing and storing, wherein the freezing density is 1 multiplied by 107One cell/mL of the frozen stock solution.
Comparative example 1
A kit for culturing liver-derived stem cells is provided with a plurality of reagent bottles, wherein a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the reagent bottles, and the culture medium additive comprises the following components in parts by weight:
dextran 5g
Calcium pantothenate 0.6g
Sodium starch phosphate. 0.6 g.
Comparative example 2
A kit for culturing liver-derived stem cells is provided with a plurality of reagent bottles, wherein a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the reagent bottles, and the culture medium additive comprises the following components in parts by weight:
comparative example 3
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
0.5g of glycine.
Comparative example 4
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
synephrine 0.5g
Xylitol 0.5 g.
Comparative example 5
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
hydroxypropyl cyclodextrin 0.6 g.
Comparative example 6
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
sodium carboxymethylcellulose 0.6g
Starch glycolate 0.2 g.
Comparative example 7
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
0.15g of sodium citrate.
Comparative example 8
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the culture medium additive also comprises the following components in parts by weight:
p-aminobenzoic acid 0.3g
0.15g of potassium sorbate.
Comparative example 9
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the frozen stock solution is mainly prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide and chitosan oligosaccharide with water for injection, wherein each L of the water for injection dissolves 40g of DMEM/F12 dry powder culture medium, 5g of dimethyl sulfoxide and 5g of chitosan oligosaccharide.
Comparative example 10
A kit for culturing liver-derived stem cells, which is different from example 3 in that: the frozen stock solution is mainly prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, lactosan and benzalkonium chloride in water for injection, wherein each L of the water for injection is used for dissolving 40g of DMEM/F12 dry powder culture medium, 5g of dimethyl sulfoxide, 5g of lactosan and 0.3g of benzalkonium chloride.
Comparative example 11
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contained 4g of methylcellulose.
Comparative example 12
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contains polyvinyl alcohol and calcium glycerophosphate, wherein 4g of the polyvinyl alcohol and 0.5g of the calcium glycerophosphate are dissolved in each L of the water for injection.
Comparative example 13
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contains 0.3g of sodium stearate lactate.
Comparative example 14
A kit for culturing liver-derived stem cells, which is different from example 11 in that: the frozen stock solution also contains sodium stearate lactate and sodium propionate, wherein 0.3g of sodium stearate lactate and 0.2g of sodium propionate are dissolved in each L of the water for injection.
Analysis of the Effect of Medium additives on the proliferation Capacity of complete Medium
The total culture media prepared by the culture media additives provided in examples 3, 5 and 1-4 were prepared according to the method of example 16, and the total culture media were cultured and expanded according to the method of example 16, and then the cells were counted by trypan blue classical staining method, and the number of primary and 3 rd generation live liver stem cells were counted, respectively, and the results are shown in table 1.
TABLE 1 evaluation of the in vitro culture amplification Effect of hepatic Stem cells
From the above results, it can be seen that the proliferation rate of the liver-derived stem cells cultured in the complete medium prepared by using the medium additive provided in example 3 is significantly higher than that of the cell culture solution of CN104928238A, and is also better than that of the complete medium prepared by using the medium additives of comparative examples 1 and 2, which indicates that the medium additive provided in the present invention can improve the proliferation ability of the complete medium prepared by using the medium additive.
The proliferation rate of the liver-derived stem cells cultured by the complete medium prepared by the medium additive provided in example 5 is better than that of the liver-derived stem cells cultured by the complete medium prepared by the medium additives provided in example 3, comparative example 3 and comparative example 4, which indicates that the addition of synephrine and glycine to the medium additive is beneficial to improving the proliferation capacity of the complete medium prepared by the medium additive.
Analysis of the Effect of Medium additives on complete Medium stability
Complete media were prepared under sterile conditions by the method of example 16 using the medium additives of example 3, example 7, comparative example 5 and comparative example 6, and the complete media were subjected to accelerated stability test and room temperature test:
1. accelerated test
Placing the complete culture medium at 40 +/-2 ℃ and 75 +/-5% of relative humidity for 6 months, sampling once at the end of 1 month, 2 months, 3 months and 6 months during the test period, and detecting the property, color and smell of the complete culture medium, wherein the results show that all indexes of the complete culture medium prepared by adopting the culture medium additive provided in example 7 have no obvious change; in contrast, the complete media prepared by the media additives provided in example 3, comparative example 5 and comparative example 6 exhibited a phenomenon of darkening in color.
2. Long term test
Placing the complete culture medium at 25 ℃ +/-2 ℃ and under the condition that the relative humidity is 60% +/-10% for 12 months, respectively sampling once at the end of 0 month, 3 months, 6 months, 9 months and 12 months during the test period, and detecting the character, color and smell of the complete culture medium, wherein the results show that all indexes of the complete culture medium prepared by adopting the culture medium additive provided by the embodiment 7 have no obvious change; in contrast, the complete media prepared by the media additives provided in example 3, comparative example 5 and comparative example 6 showed precipitation and color deepening.
From the results of the accelerated test and the long-term test, it is known that the stability of the complete culture medium can be remarkably improved by adding hydroxypropyl cyclodextrin and sodium starch glycolate into the culture medium additive, the problems of precipitation, layering, discoloration and the like of the complete culture medium are avoided, one component is reduced, or one component is changed, so that the stability of the complete culture medium is reduced.
Analysis of Effect of Medium additives on safety of complete Medium
Complete media were prepared aseptically from the media additives of example 3, example 9, comparative example 7 and comparative example 8 by the method of example 16, and the total number of bacteria in the complete media was measured after storing the complete media in a closed state at 25 ℃. + -. 2 ℃ and a relative humidity of 60%. + -. 10% for 12 months, as shown in Table 2.
TABLE 2 Total bacteria count in complete Medium
| Example 3 | Example 9 | Comparative example 7 | Comparative example 8 | |
| Total number of bacteria (cfu/mL) | 897 | 53 | 863 | 838 |
From the above results, it can be seen that the addition of p-aminobenzoic acid and sodium citrate to the medium additive can effectively improve the ability of the complete medium to prevent bacterial infection, effectively inhibit the growth and reproduction of bacteria in the complete medium, ensure the safe use of the complete medium, and reduce one of the components or change one of the components, which can lead to the reduction of the antibacterial ability of the complete medium.
Evaluation of influence of frozen stock solution on freezing and storing performance of hepatic stem cells
The liver-derived stem cells were added to 1.5mL of the frozen stocks provided in examples 11, 9 and 10 and the cell frozen stock provided in CN104928238A, respectively, and resuspended in 2mL of a cell freezing tube, and the number of cells was controlled to 1X 106Per mL; quickly transferring the cell freezing tube to a cell program cooling box added with isopropanol, putting the cell freezing tube into an ultra-low temperature refrigerator at minus 80 ℃ for 2-3 days, and transferring the cell freezing tube to liquid nitrogen for preservation; after one month, cell recovery is carried out, the cell cryopreserving tube stored in liquid nitrogen is taken out, the cell cryopreserving tube is transferred to a water bath kettle at 37 ℃ for quick thawing, the cells are transferred to 10mL of complete culture medium prepared by the method of the embodiment 18 by a pipette, after the cells are lightly blown and uniformly mixed, 0.5mL of cell suspension is taken to detect the cell survival rate and the cell morphological change, and the experimental result is shown in Table 3.
TABLE 3 Effect of cryopreservation solution on cryopreservation Performance of hepatic Stem cells
Comparing the freezing performance of the freezing solution provided in example 11 with the freezing performance of the freezing solution provided in comparative examples 9 and 10 and the cell freezing solution provided in CN104928238A, it can be seen that the freezing solution provided in the present invention has relatively better freezing performance, and the survival rate of the liver-derived stem cells after recovery is higher, and is more beneficial to maintaining and stabilizing the cell morphology before and after the liver-derived stem cells are frozen.
Frozen stock solution stability evaluation
1. Accelerated test
Taking the frozen stock solutions provided by the embodiment 11, the embodiment 13, the comparison 11 and the comparison 12, placing the frozen stock solutions at the temperature of 40 +/-2 ℃ and the relative humidity of 75 +/-5% for 6 months, respectively sampling once at the end of 1 month, 2 months, 3 months and 6 months during the test period, and detecting the properties, the color and the smell of the frozen stock solutions, wherein the results show that all indexes of the frozen stock solutions provided by the embodiment 13 have no obvious change; while the frozen stocks provided in example 11, comparative example 11 and comparative example 12 showed a small amount of precipitation and became darker in color.
2. Long term test
Taking the frozen stock solutions provided by the example 11, the example 13, the comparative example 11 and the comparative example 12, placing the frozen stock solutions at the temperature of 25 +/-2 ℃ and the relative humidity of 60 +/-10% for 12 months, respectively sampling once at the end of 0 month, 3 months, 6 months, 9 months and 12 months during the test period, and detecting the properties, the color and the smell of the frozen stock solutions, wherein the results show that all indexes of the frozen stock solutions provided by the example 13 have no obvious change; in contrast, the frozen stocks provided in examples 11, 11 and 12 were precipitated and darkened.
From the results of the above accelerated test and the long-term test, it is found that the stability of the frozen liquid can be remarkably improved by adding methylcellulose and calcium glycerophosphate to the frozen liquid, and the stability of the frozen liquid can be reduced by reducing one of the components or changing one of the components.
Evaluation of safety of frozen stock solution
The total bacterial count in the frozen stocks obtained in example 11, example 15, comparative example 13 and comparative example 14 were determined by storing the respective stocks at 25 ℃. + -. 2 ℃ and 60%. + -. 10% relative humidity for 12 months under sealed conditions, and the results are shown in Table 4.
TABLE 4 Total bacteria count in frozen stock
| Example 11 | Example 15 | Comparative example 13 | Comparative example 14 | |
| Total number of bacteria (cfu/mL) | 899 | 162 | 871 | 843 |
From the results, the addition of the sodium stearate lactate and the potassium sorbate to the frozen stock solution can improve the antibacterial capacity of the frozen stock solution, effectively inhibit the breeding and reproduction of bacteria in the frozen stock solution, and ensure the use safety of the frozen stock solution.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered by the claims of the present invention.
Claims (7)
1. The kit for culturing the liver-derived stem cells is characterized in that a plurality of reagent bottles are arranged in the kit, a culture solution, a culture medium additive, a growth factor I, a growth factor II, a digestive juice, a cleaning solution, a frozen stock solution and cell seeds are respectively contained in the plurality of reagent bottles, the culture solution is a DMEM/F12 culture medium solution, the growth factor I is bFGF, the growth factor II is EGF, the digestive juice is pancreatin, the cleaning solution is a PBS buffer solution, the cell seeds are the liver-derived stem cells, and the culture medium additive comprises the following components in parts by weight:
the frozen stock solution is prepared by dissolving DMEM/F12 dry powder culture medium, dimethyl sulfoxide, chitosan oligosaccharide and benzalkonium chloride in water for injection, wherein each L of the water for injection is used for dissolving 30-50g of the DMEM/F12 dry powder culture medium, 3-8g of the dimethyl sulfoxide, 3-6g of the chitosan oligosaccharide and 0.2-0.5g of the benzalkonium chloride.
2. The kit according to claim 1, wherein the medium additive further comprises the following components in parts by weight:
synephrine 0.3-1
0.1-0.7 of glycine.
3. The kit according to claim 1, wherein the medium additive further comprises the following components in parts by weight:
hydroxypropyl cyclodextrin 0.3-0.8
Starch sodium glycolate 0.1-0.5.
4. The kit according to claim 1, wherein the medium additive further comprises the following components in parts by weight:
p-aminobenzoic acid 0.2-0.5
0.1-0.2 of sodium citrate.
5. The kit according to claim 1, wherein the frozen stock solution further comprises methylcellulose and calcium glycerophosphate, and wherein 2 to 5g of the methylcellulose and 0.2 to 0.8g of the calcium glycerophosphate are dissolved in each L of the water for injection.
6. The kit according to claim 1, wherein the frozen stock solution further contains sodium lactate stearate and potassium sorbate, and 0.2 to 0.4g of the sodium lactate stearate and 0.1 to 0.3g of the potassium sorbate are dissolved in each L of the water for injection.
7. A method for culturing hepatic stem cells by using the kit of any one of claims 1 to 6, wherein the method comprises the following steps:
s1, mixing a culture solution, a culture medium additive, a growth factor I and a growth factor II according to a weight ratio of 3: 0.01: 1: 1, preparing a complete culture medium, and storing the complete culture medium in a refrigerator at 4 ℃;
s2, adding the cell seeds into the cleaning solution, centrifuging for 5min at 4 ℃ and 1000rpm, collecting cells, adding the collected cells into the complete culture medium provided in the step S1 for re-suspension, and re-suspending according to the proportion of 1 × 106-2×106The cells/mL were inoculated in culture flasks at 37 ℃ with 5% C02In the environment of (2), hatching cells by using a standard incubator, and replacing the complete culture medium every 24 hours in the hatching process;
s3, when the cell growth area reaches 85-95%, cleaning for 2 times by using a cleaning solution, adding 5% of digestive juice, appropriately shaking and shaking the culture bottle to enable the digestive juice to be fully contacted with the cells, then putting the cells into an incubator to be digested for 1-2min, finally adding a complete culture medium preheated to 37 ℃ to stop digestion, and then carrying out passage on the obtained cells according to the proportion of 1: 3;
s4, repeating the step S3 for 1 time, then centrifuging for 5min at 4 ℃ and 1000rpm, repeating the centrifugation for 2 times, then collecting cells, putting the cells into a freezing medium for freezing and storing, wherein the freezing density is 1 multiplied by 107-2×107One cell/mL of the frozen stock solution.
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| CN102154210A (en) * | 2010-12-28 | 2011-08-17 | 沈达青 | Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof |
| CN104531611A (en) * | 2014-12-29 | 2015-04-22 | 中国人民解放军第二军医大学 | Specificity surface molecule marker CD63 of hepatic stem cell and application thereof |
| CN105807063A (en) * | 2014-12-29 | 2016-07-27 | 中国人民解放军第二军医大学 | Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases |
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| CN102154210A (en) * | 2010-12-28 | 2011-08-17 | 沈达青 | Method for preparing hepatic stem cell decorated by insulin gene and application and preparation thereof |
| CN104531611A (en) * | 2014-12-29 | 2015-04-22 | 中国人民解放军第二军医大学 | Specificity surface molecule marker CD63 of hepatic stem cell and application thereof |
| CN105807063A (en) * | 2014-12-29 | 2016-07-27 | 中国人民解放军第二军医大学 | Use of CD63 in preparation of kit for liver disease diagnosis or drug for preventing or treating liver diseases |
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