CN115968862A - Cell cryopreservation liquid and application thereof - Google Patents
Cell cryopreservation liquid and application thereof Download PDFInfo
- Publication number
- CN115968862A CN115968862A CN202211678651.7A CN202211678651A CN115968862A CN 115968862 A CN115968862 A CN 115968862A CN 202211678651 A CN202211678651 A CN 202211678651A CN 115968862 A CN115968862 A CN 115968862A
- Authority
- CN
- China
- Prior art keywords
- cell
- mesenchymal stem
- stem cells
- cryopreservation
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a cell cryopreservation solution and application thereof, wherein the cryopreservation solution consists of a basic culture medium, DMSO-DEX40 and human serum albumin, wherein the mass ratio of the DMSO-DEX40 in the cell cryopreservation solution is 5-25%. The invention has stable preservation effect on the mesenchymal stem cells by optimizing the component composition of the cell cryopreservation solution, and particularly has high and stable cryopreservation recovery survival rate on the umbilical cord mesenchymal stem cells. The cell freezing solution avoids the risk of introducing animal-derived viruses, has simple and definite components, can stably obtain higher cell recovery survival rate, and has low application risk after cell recovery.
Description
Technical Field
The invention relates to the technical field of cell preservation, in particular to a cell cryopreservation solution and application thereof.
Background
The aim of cell cryopreservation is to slow down the metabolic activity of cells and maintain life in a low-temperature environment of liquid nitrogen, ice crystal formation is one of the main causes of cell death in the freezing process of cell suspension, and the slow cooling rate is kept to avoid the ice crystal formation so that the cryopreservation protective agent gradually enters the interior of the cells to form a concentration gradient. When the cooling is continuously carried out, the water in the cells gradually flows out of the cells under the osmotic pressure of the freezing agent, and the cells are completely dehydrated and shrunk to reach the optimal freezing state when the concentration of the freezing agent in the cells reaches the maximum. If the freezing rate is too fast, the water in the cells cannot be completely discharged, so that ice crystals in the cells are formed and the cells are damaged. However, the freezing medium has a certain toxic effect on cells, and the exposure time of the cells in the freezing medium is too long due to the low freezing speed, so that the cells are poisoned. Both the cytotoxic effects of cryoprotectants and cellular damage due to intracellular ice crystals are widely recognized, so cryopreservation of cells requires adequate cooling rates to ensure that the cells are subjected to minimal cryoprotectant toxicity and ice crystal damage when the cryopreservation temperature is reached. Different cell types have different membrane permeability parameters, and therefore the optimal cryopreservation rate is also different.
In addition to the freezing rate, cryoprotectants, which are classified into osmotic and non-osmotic protectants according to their ability to cross cell membranes, are also one of the important factors to be considered for reducing freezing damage, and are mainly used to prevent the formation of ice crystals within cells, to avoid the toxicity of high concentrations of cryoprotectants, and to dehydrate cells within tolerance, among others. The protective capacity of osmotic protectants is a highly soluble water-soluble complex composed of many small molecules that can depress the freezing point of water at high concentrations, thereby reducing the formation of large ice crystals during freezing. Non-osmotic cryoprotectants are trehalose, sucrose, lactose, glucose, mannitol, sorbitol, polymeric hydroxyethyl starch and polyvinylpyrrolidone, which are capable of protecting cells at lower concentrations, but require higher freezing rates, e.g., polymeric hydroxyethyl starch cannot penetrate to cause an increase in extracellular concentration, and form reverse osmosis at low temperatures to effect dehydration for protection. In other words, the concentration of the non-penetrating cryoprotectant outside the cells results in dehydration of the cells primarily during the initial stages of freezing. The protective effect of trehalose is the interaction with lipid membranes, which maintains protein stability during cryopreservation and resuscitation, and forms a vitrified matrix to inhibit intracellular ice crystal generation, but even an impermeable protective agent can cause cell damage. The development of more effective cryopreservation agents is also one of the challenges in cell cryopreservation, and studies have been made to focus on the mechanism of toxicity of the impermeable protective agent to cells in order to minimize its toxicity.
At present, the conventional cryopreservation method of Mesenchymal Stem Cells (MSCs) in a laboratory adopts a basic culture medium containing FBS and DMSO, also adopts a cryopreservation solution containing 5% or 10% DMSO and human serum albumin, and then the cryopreservation solution is put into liquid nitrogen or a liquid nitrogen gas phase after being cooled from room temperature to minus 80 ℃ at a certain cryopreservation rate. However, the cells preserved in the FBS-containing cryopreservation solution have the risk of virus of a heterogeneous animal source in clinical application, and the cell cryopreservation solution prepared by two components of FBS and DMSO, which is conventional in a laboratory, has higher cryopreservation cost due to higher content of FBS. The use of a frozen stock solution containing DMSO and human serum albumin is an improvement over the method used to freeze hematopoietic stem cells and lymphocytes, and may not be the best method for freezing MSCs. The reduction of cell damage during cell cryopreservation is not only dependent on cell suspension or adhesion clustering, but also has a very important relationship with the source of the cells. Studies have shown that the survival rate of cryopreserved MSCs from different tissues is also significantly different, e.g., the survival rate of MSCs isolated from adipose tissue is higher than that isolated from pulp and bone marrow. Therefore, the research on the physical and biological processes after the MSCs are frozen and thawed is the basis for optimizing the freezing and preserving method so as to ensure that the MSCs maintain the structural integrity and the functional characteristics.
Disclosure of Invention
In view of the above, the invention provides a cell cryopreservation solution and an application thereof, the cell cryopreservation solution has simple components, has a good cryopreservation effect on umbilical cord mesenchymal stem cells, and can obtain a high cell viability rate and stable data after cell recovery.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a cell cryopreservation solution, which consists of a basic culture medium, DMSO-DEX40 and human serum albumin, wherein the mass ratio of the DMSO-DEX40 in the cell cryopreservation solution is 5% -25%.
Further, the basic medium consists of DMEM/F12 and PALL Ultroser TM G is prepared byWherein PALL Ultroser TM The mass ratio of G in the basic culture medium is 2-4%.
Further, PALL Ultroser TM The mass ratio of G in the basal medium is 2%.
Further, the cell freezing solution comprises the following components in percentage by weight: DMSO-DEX40,5% -20%; 10% -20% of human serum albumin; basic culture medium and the balance.
Preferably, the cell cryopreservation solution comprises the following components in percentage by weight: DMSO-DEX40, 20%; 10% of human serum albumin; basic culture medium, the rest; PALL Ultroser TM The mass ratio of G in the basal medium is 2%.
Preferably, the cell cryopreservation solution comprises the following components in percentage by weight: DMSO-DEX40, 10%; 20% of human serum albumin; basic culture medium, the rest; PALL Ultroser TM The mass ratio of G in the basal medium is 2%.
Further, the preparation method of the frozen stock solution comprises the following steps: mixing all the components, and storing at 2-8 deg.C.
In a second aspect, the invention provides an application of the cell cryopreservation solution in mesenchymal stem cell preservation.
Further, the mesenchymal stem cell comprises: bone marrow, dental pulp, adipose, umbilical cord derived mesenchymal stem cells.
Preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
In a third aspect, the present invention provides a cryopreservation method of mesenchymal stem cells, comprising the following steps:
step 1, carrying out adherent culture on mesenchymal stem cells until the confluence degree reaches 85% -90%, collecting culture supernatant of the mesenchymal stem cells, cleaning the adherent mesenchymal stem cells by using a proper amount of normal saline, and removing a cleaning solution;
step 2, adding the mesenchymal stem cells into a digestive juice for digestion, and then adding the mesenchymal stem cell culture supernatant collected in the step 1 to terminate digestion;
step 3, collecting and centrifuging the cell suspension after digestion is ended, discarding the upper liquid, resuspending the precipitate with physiological saline, and sampling for cell counting;
step 4, centrifuging the sampled cell suspension again, discarding the upper liquid, and adding the cell frozen stock solution according to the cell counting result;
step 5, transferring the cell suspension added with the cell cryopreservation liquid into a cell cryopreservation tube, sealing the cell cryopreservation tube, putting the cell cryopreservation tube into a program cooling box added with isopropanol, and transferring the program cooling box into a temperature system at-80 ℃;
and 6, transferring the cell freezing tube to an environment system at the temperature of not higher than-135 ℃ after 16h, and sealing.
Further, in the step 2, the preparation method of the digestive juice comprises the following steps: and (3) diluting 0.25 mass percent of Trypsin-EDTA by 1 time by using physiological saline to obtain the Trypsin-EDTA.
Further, in the step 4, the density of the cells in the cell freezing medium is 2X 10 6 ～1×10 7 Each/ml.
In a fourth aspect, the present invention provides a method for improving the activity of a cryopreserved resuscitated cell of a mesenchymal stem cell, comprising:
step one, freezing and storing the mesenchymal stem cells by adopting the freezing and storing method;
preparing a water bath environment at 37 +/-0.1 ℃ and a basic culture medium at room temperature before resuscitation;
step three, taking out the frozen mesenchymal stem cells, and placing the frozen mesenchymal stem cells in a water bath environment at 37 +/-0.1 ℃ to convert the frozen mesenchymal stem cells from a solid state into a cell suspension;
transferring the cell suspension to a basic culture medium at room temperature, centrifuging, and discarding the supernatant;
and step five, adding a basal medium for re-resuspending.
Further, the basic medium consists of DMEM/F12 and PALL Ultroser TM G, wherein PALL Ultroser TM The mass ratio of G in the basic culture medium is 2-4%.
The invention has the beneficial effects that:
the cell cryopreservation solution with stable preservation effect on the mesenchymal stem cells is formed by optimizing the component composition of the cell cryopreservation solution, and particularly the cell cryopreservation solution has high and stable cryopreservation recovery activity rate on the umbilical cord mesenchymal stem cells. The cell freezing solution avoids the risk of introducing animal-derived viruses, has simple and definite components, can stably obtain higher cell recovery survival rate, and has low application risk after cell recovery.
Drawings
FIG. 1 is a photograph of umbilical cord mesenchymal stem cells numbered UC005 in example 1 of the present invention cultured under 40 times of the lens (Panel A) and 100 times of the lens (Panel B) until the 5 th generation confluency reaches 90%.
FIG. 2 is a photograph of umbilical cord mesenchymal stem cells of present invention, numbered UC005, at 40 times under the microscope (panel A) and 100 times under the microscope (panel B), marked as 6 th generation after 5 th generation resuscitation adherence.
Detailed Description
The DMSO-DEX40 used in the examples of the invention was obtained from WAK CHEMIE, germany, containing 55g/dl DMSO USP Grade and 5g/dl Dextran 40USP Grade.
Human Serum Albumin (HSA) used in the examples of the present invention was purchased from Jetbelin.
Trypsin-EDTA adopted in the embodiment of the invention is purchased from gibco.
DMEM/F12 used in the examples of the present invention was purchased from gibco.
Ultroser adopted by embodiment of the invention TM G was purchased from Pall corporation.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The invention provides a cell freezing medium, which consists of a basic medium, DMSO-DEX40 and human serum albumin, wherein the basic medium consists of DMEM/F12 and PALL Ultroser TM G, wherein PALL Ultroser TM The volume ratio of G in the basic culture medium is 2-4%, and DMSO-DEX40 is in frozen stock solutionThe mass ratio is 5-25%. The basal medium is abbreviated hereinafter as M4.
In some embodiments of the present invention, the cell cryopreservation solution comprises the following components by weight: DMSO-DEX40,5% -20%; 10% -20% of human serum albumin; basic culture medium and the balance.
In some preferred embodiments of the present invention, the cell cryopreservation solution comprises the following components by weight: DMSO-DEX40, 20%; 10% of human serum albumin; basic culture medium and the balance. Or the cell freezing solution comprises the following components in percentage by weight: DMSO-DEX40, 10%; 20% of human serum albumin; basic culture medium and the balance. In both preferred modes, PALL Ultroser TM The mass ratio of G in the basal medium is 2%.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The embodiment provides different cryopreservation liquid formulas for performing cryopreservation recovery experiments on umbilical cord mesenchymal stem cells of the 5 th generation, and the cryopreservation recovery experiments specifically comprise the following steps:
1) Experimental materials:
umbilical cord mesenchymal stem cell samples were collected at dendran (hangzhou) hospital at 3 months in 2022, screened for 7 pathogens according to national standards, and evaluated by the ethical committee of hospitals to meet the collection standards, and umbilical cord tissues of newborn were cultured to 5 th generation cells, and the experimental protocol had 2 donors, with the cell codes of UC005-P5 and UC009-P5.
The specific method of culturing cells from umbilical cord tissue of all the 2 donors to passage 5 is as follows:
1. sample processing and tissue extraction
1-1, shearing part of umbilical cord tissues (6-8 cm) in a good state in an umbilical cord collecting box, and transferring the sheared umbilical cords into a sterilizing tray.
1-2, washing 2 times with saline (removing a large amount of microorganisms and extravasated blood on the surface), adding 50ml of 75% alcohol into a bottle, and disinfecting and washing the umbilical cord surface.
1-3, washing the umbilical cord with saline water for 2 times again (washing off alcohol residues), cutting the umbilical cord into small segments of 2cm-3cm, and if the umbilical cord still has blood stasis, squeezing out the saline water with toothed forceps to wash.
1-4, taking a small section of umbilical cord, cutting the umbilical cord along the texture of the external surface skin by using scissors, carefully tearing the external surface skin of the umbilical cord by using toothed forceps and spreading the umbilical cord open, and removing venous blood vessels and arterial blood vessels in the umbilical cord.
1-5, taking Walton's glue: using a pair of toothed forceps to pick out the Walton gel in the umbilical cord, placing the Walton gel in another sterile vessel, and paying attention not to puncture the epidermis.
1-6, transferring the peeled Wharton's jelly into a 50ml centrifugal tube, adding a small amount of saline, and shearing the Wharton's jelly to the size of rice grains by using sterile surgical scissors. Adding appropriate amount of saline water to constant volume, and centrifuging and washing for 2 times at 300g for 6 min.
1-7, adding 5ml of M4 and about 1ml of sheared tissue seed bottles into each T75 bottle according to the tissue precipitation amount after centrifugation, slightly and uniformly shaking to uniformly spread the Wharton jelly particles on the bottom surface of the culture bottle, and marking the cell number, the generation number (P0), the date and the operator.
1-8, placing the culture bottle with the Wharton's jelly granules in an inclined way, standing for adherence for 20-30min, and then placing the culture bottle into a cell culture box with the temperature of 37 ℃ and the carbon dioxide concentration of 5% for standing culture.
2. Cell amplification and collection
2-1, replacing the Wharton's jelly particles cultured for 2-3 days with a new culture solution, sucking the old culture solution by using a 10ml pipette, discarding the old culture solution, sucking 5ml of the new culture solution by using a pipette, adding the new culture solution into a culture bottle, covering the bottle tightly, and marking the cell number, generation number, date and operator.
2-2, replacing the Wharton's jelly particles cultured for 6-7 days with a new culture solution, sucking the old culture solution by using a 10ml pipette, discarding the old culture solution, sucking 5ml of the new culture solution by using a pipette, adding the new culture solution into a culture bottle, covering the bottle tightly, and marking the cell number, generation number, date and operator.
2-3, observing the Wharton jelly particles cultured for 10-14 days under a microscope, slightly beating the culture bottle after the cells grow out around the Wharton jelly particles to enable the Wharton jelly particles to fall off, discarding the fallen particles and the old culture solution, adding 7ml of new culture solution, and marking the cell number, generation, date and operator.
2-4, when the cells are fully paved on about 80-90% of the bottom of the culture bottle, carrying out trypsinization passage, sucking out the culture solution by a pipette to discard, slightly washing the cells by saline, adding 2-3 ml of 0.25% trypsin, flatly laying the culture bottle, and slightly shaking to ensure that the trypsin is paved on the bottom of the culture bottle. The vessel walls were gently tapped while viewing under an inverted microscope, and digestion was stopped with complete medium when most of the cells began to come out of the walls. Transferring the digested cell suspension into a 50ml centrifugal tube through a filtering membrane with the diameter of 70um, washing the culture bottle with a small amount of saline water, transferring the culture bottle into the centrifugal tube through the filtering membrane with the diameter of 70um, 300g,6min for centrifugal washing for 2 times, pouring off supernatant to a waste liquid tank, resuspending cells with a proper amount of culture solution, adding the culture solution to a constant volume of 50ml, transferring the cells into 2T 175 culture bottles respectively, shaking the culture bottles lightly and uniformly to enable the cells to be uniformly paved on the bottom surface of the culture bottle, and marking the cell number, the generation number (P1), the date and an operator.
2-5, when the cells are fully paved on about 90% of the bottom of the culture bottle, collecting and freezing the cells, sucking out the culture solution by a pipette to discard, slightly washing the cells by saline, adding 3ml of saline and 3ml of 0.25% trypsin respectively, flatly placing the culture bottle and slightly shaking the culture bottle to enable the trypsin to be paved on the bottom of the culture bottle. The flask wall was gently tapped while viewing under an inverted microscope, and digestion was stopped with complete medium when most of the cells began to detach from the wall. Transferring the digested cell suspension into a 50ml centrifuge tube, washing a culture flask with a small amount of saline, transferring into the centrifuge tube, centrifuging for 300g and 6min, pouring out the supernatant to a waste liquid tank, sucking more than 10ml of saline to resuspend cells by using a liquid transfer gun, sampling, inspecting (quantity and activity), adding saline to fix the volume to 50ml, centrifuging for 300g and 8 min. Determining the number of subcultured bottles according to the number of cells, according to 1 x 10^ 6 cells were passaged at the seed vial density of each T175 vial, the cells were labeled P2, and the residual cell suspension was sampled for examination (bacteria, fungi, endotoxin, mycoplasma).
2-6, repeating the steps 2-5 to expand the cells from P2 to P5. Wherein the cells are subjected to detection of bacteria, fungi, mycoplasma and endotoxin at the 1 st generation collection, the 3 rd generation collection and the 5 th generation collection.
Figure 1 shows photographs of umbilical cord mesenchymal stem cells numbered UC005 cultured under 40-fold mirror (panel a), 100-fold mirror (panel B) until the 5 th passage confluency reaches 90%. Figure 2 gives photographs of umbilical cord mesenchymal stem cells numbered UC005 under 40-fold microscopy (panel a), marked as passage 6 after 5 th resuscitation attachment (panel B). Shows that the umbilical cord mesenchymal stem cells have better cell activity before and after cryopreservation.
Obtaining 7 groups of 5 th generation cell samples of the umbilical cord mesenchymal stem cells from samples of each donor according to the same culture method, wherein the sample numbers are 1-7 respectively;
2) The formulation of the frozen stock solution is shown in table 1.
TABLE 1 cell cryopreservation liquid formula corresponding to different sample numbers
| Sample number | Cell freezing solution formula (weight percentage composition) |
| 1 | DMSO-DEX40，10％+M4，90％ |
| 2 | DMSO-DEX40,5% + human serum albumin, 5% + M4, 90% |
| 3 | DMSO-DEX40,5% + human serum albumin, 10% + M4, 85% |
| 4 | DMSO-DEX40, 10% + human serum albumin, 5% + M4, 85% |
| 5 | DMSO-DEX40, 10% + human serum albumin, 10% + M4, 80% |
| 6 | DMSO-DEX40, 10% + human serum albumin, 20% + M4, 70% |
| 7 | DMSO-DEX40, 20% + human serum albumin, 10% + M4, 70% |
PALL Ultroser in Table 1 TM The mass ratio of G in M4 is 2%.
3) Performing cryopreservation and recovery experiments on the umbilical cord mesenchymal stem cells on the 7 cell samples obtained in the step 2) by adopting respective corresponding cell cryopreservation liquid, and then detecting the survival rate, wherein the method specifically comprises the following steps:
step 1, collecting the 5 th generation mesenchymal stem cell culture supernatant of each cell sample, cleaning the adherent mesenchymal stem cells with a proper amount of normal saline for 2 times, and discarding the cleaning solution.
Step 2, adding the mesenchymal stem cells into digestive juice for digestion; the preparation method of the digestive juice comprises the following steps: and (3) diluting 0.25 mass percent of Trypsin-EDTA by 1 time by using physiological saline to obtain the Trypsin-EDTA. And (3) observing the digestion process of adherent cells by using a microscope, and stopping the digestion process by using the culture supernatant collected in the step (1) when the cells are in a rounded state and the cells can fall off by lightly tapping the bottle wall.
And 3, collecting all cell suspensions after digestion termination into a 50ml centrifuge tube, and centrifuging for 300g and 6 min. The supernatant was discarded, the pellet resuspended in saline, and the sample taken for cell counting.
Step 4, the sampled cell suspension is centrifuged again for 300g and 6 min. The supernatant was discarded, and cell culture medium was added according to the cell count result. The cell freezing density is 2 multiplied by 10 6 The volume of the powder is 0.5ml.
And 5, transferring the cell suspension added with the freezing storage solution into a 1.8ml cell freezing storage tube, capping the freezing storage tube, wrapping the freezing storage tube by a sealing film, and transferring the freezing storage tube into a cell program cooling box (isopropanol is added into the box, so that programmed cooling can be realized, and damage to cells caused by rapid cooling is avoided), wherein the program cooling box is placed under the environment condition of-80 ℃.
And 6, after 16 hours, transferring the cells in the programmed cooling box to a liquid nitrogen phase at the temperature of not higher than-135 ℃ or a meteorological environment for about 1 month.
And 7, preparing a water bath environment at 37 +/-0.1 ℃ before resuscitation. And M4 in 50ml centrifuge tubes ready to return to room temperature.
And 8, taking out the cells from the liquid nitrogen environment, wrapping the cells by using a sterile PE glove, placing the wrapped cells in a water bath environment at 37 ℃, and continuously shaking the cell cryopreservation tube until the solid matters are just completely dissolved (changing from the solid matters to suspension matters).
Step 9, transferring the cells in the cryopreserved tube to a prepared room temperature M4, and centrifuging for 300g and 6 min. And (4) discarding the supernatant after the centrifugation is finished, re-suspending by using M4, uniformly blowing and beating (the blowing and beating times are limited to 5 times, and 7 samples are kept consistent), and sampling and checking for counting. Samples were taken from the cell primary suspension in consistent volumes, all 150ul.
And step 10, detecting the cell viability of the sampled sample. The operation is as follows: the cell suspension in the ep tube was blown up 10 times, and 20ul was taken. 20ul of 0.4% trypan blue stain was taken, mixed and beaten 10 times. From a suspension of 0.4% trypan blue and cells in equal volume ratio, 20ul was added to an automatic cell counting plate and the instrument was operated for number and viability. The instrument is a Countstar BioTech automated cell counter. And finally, carrying out corresponding statistics on the counting activity rate and the number displayed by the instrument. The results of the experiment are shown in tables 2 and 3:
TABLE 2 Experimental results of 7 samples of UC005-P5
TABLE 3 Experimental results of 7 samples of UC009-P5
The data in tables 2 and 3 show that the partial replacement of DMSO-DEX40 with 5% HSA in the cell culture medium (sample 2) did not significantly increase the viability of the cells, but with the increase of the proportion of HSA in the culture medium (samples 4 to 7), the viability of the cells was significantly increased, and the viability of the cells was the highest for the culture medium with DMSO-DEX40 proportion of 10% to 20% HSA or with DMSO-DEX40 proportion of 20% to 10% HSA, and the difference between the viability of the two was small.
Example 2
The embodiment provides a cryopreservation recovery experiment aiming at different mesenchymal stem cells by different cryopreservation liquid formulas, which specifically comprises the following steps:
1) Experimental materials:
umbilical cord mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells of the same source as in example 1, and the cells are coded into UC005-P5 umbilical cord mesenchymal stem cells;
bone marrow mesenchymal stem cell source: from BALB/C mice.
Umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells are cultured to the 5 th generation by the method of example 1, and the umbilical cord mesenchymal stem cells and the bone marrow mesenchymal stem cells are cultured in 2 groups respectively in parallel for standby. Sample numbers of umbilical cord 1, umbilical cord 2, bone marrow 1 and bone marrow 2;
2) The formulation of the frozen stock solution is shown in table 4.
TABLE 4 cell cryopreservation liquid formula corresponding to different sample numbers
| Sample number | Cell freezing solution formula (weight percentage composition) |
| Umbilical cord 1 | DMSO-DEX40, 10% + human serum albumin, 20% + M4, 70% |
| Umbilical cord 2 | DMSO-DEX40, 20% + human serum albumin, 10% + M4, 70% |
| Bone marrow 1 | DMSO-DEX40, 10% + human serum albumin, 20% + M4, 70% |
| Bone marrow 2 | DMSO-DEX40, 20% + human serum albumin, 10% + M4, 70% |
PALL Ultroser in Table 1 TM The mass ratio of G in M4 is 2%.
The cell cryopreservation liquid is subjected to cryopreservation and recovery experiments of umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells according to the method of example 1, then the survival rate is detected, and the test results are shown in table 5, which shows that the cell cryopreservation liquid is more suitable for cryopreservation and recovery of umbilical cord mesenchymal stem cells.
TABLE 5 Experimental results for 4 samples
The data in table 5 show that the cell frozen stock solution of the invention is more suitable for the preservation of umbilical cord mesenchymal stem cells, but can also be used for the preservation of bone marrow mesenchymal stem cells.
In conclusion, the cell cryopreservation solution is suitable for cryopreservation of mesenchymal stem cells, and particularly has high and stable survival rate of cryopreservation recovery of umbilical cord mesenchymal stem cells. The cell freezing solution avoids the risk of introducing animal-derived viruses, has simple and definite components, can stably obtain higher cell recovery survival rate, and has low application risk after cell recovery.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A cell cryopreservation solution, which is characterized in that: the culture medium consists of a basic culture medium, DMSO-DEX40 and human serum albumin, wherein the mass ratio of the DMSO-DEX40 in a cell freezing medium is 5-25%.
2. The cell cryopreservation solution of claim 1, wherein: the basic culture medium consists of DMEM/F12 and PALL Ultroser TM G, wherein PALL Ultroser TM The mass ratio of G in the basic culture medium is 2-4%.
3. The cell cryopreservation solution of claim 2, wherein: the cell freezing solution comprises the following components in percentage by weight: DMSO-DEX40,5% -20%; 10% -20% of human serum albumin; basic culture medium and the balance.
4. A cell cryopreservation solution according to claim 2 or 3, which isIs characterized in that: the cell freezing solution comprises the following components in percentage by weight: DMSO-DEX40, 20%; 10% of human serum albumin; basic culture medium and the balance; PALL Ultroser TM The mass ratio of G in the basal medium is 2%.
5. A cell cryopreservation solution according to claim 2 or 3 wherein: the cell freezing solution comprises the following components in percentage by weight: DMSO-DEX40, 10%; 20% of human serum albumin; basic culture medium and the balance; PALL Ultroser TM The mass ratio of G in the basic culture medium is 2%.
6. The cell cryopreservation solution of claim 1, wherein: mixing all the components uniformly after burdening, and preserving at 2-8 ℃.
7. Use of the cell cryopreservation solution of any one of claims 1 to 6 in the preservation of mesenchymal stem cells.
8. Use according to claim 7, characterized in that: the mesenchymal stem cell comprises: bone marrow, dental pulp, adipose, umbilical cord derived mesenchymal stem cells.
9. A cryopreservation method of mesenchymal stem cells is characterized by comprising the following steps: the method comprises the following steps:
step 1, performing adherent culture on mesenchymal stem cells until the confluence reaches 85-90%, collecting culture supernatant of the mesenchymal stem cells, cleaning the adherent mesenchymal stem cells by using a proper amount of normal saline, and removing a cleaning solution;
step 2, adding the mesenchymal stem cells into a digestive juice for digestion, and then adding the mesenchymal stem cell culture supernatant collected in the step 1 to terminate digestion;
step 3, collecting and centrifuging the cell suspension after digestion is ended, discarding the upper liquid, resuspending the precipitate with physiological saline, and sampling for cell counting;
step 4, centrifuging the sampled cell suspension again, discarding the upper liquid, and adding the cell cryopreservation liquid of any one of claims 1 to 6 according to the cell counting result;
step 5, transferring the cell suspension added with the cell cryopreservation liquid into a cell cryopreservation tube, sealing the cell cryopreservation tube, putting the cell cryopreservation tube into a programmed cooling box added with isopropanol, and transferring the programmed cooling box into a temperature system at minus 80 ℃;
and 6, transferring the cell freezing tube to an environment system at the temperature of not higher than-135 ℃ after 16h, and sealing.
10. A method for improving the activity of cryopreserved resuscitated cells of mesenchymal stem cells is characterized by comprising the following steps: the method comprises the following steps:
step one, freezing and storing the mesenchymal stem cells by adopting the freezing and storing method of claim 9;
preparing a water bath environment at 37 +/-0.1 ℃ and a basic culture medium at room temperature before resuscitation;
step three, taking out the frozen mesenchymal stem cells, and placing the frozen mesenchymal stem cells in a water bath environment at the temperature of 37 +/-0.1 ℃ to convert the frozen mesenchymal stem cells from a solid state into a cell suspension;
transferring the cell suspension to a basic culture medium at room temperature, centrifuging, and discarding the supernatant;
and step five, adding a basal medium for re-resuspending.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211678651.7A CN115968862A (en) | 2022-12-26 | 2022-12-26 | Cell cryopreservation liquid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211678651.7A CN115968862A (en) | 2022-12-26 | 2022-12-26 | Cell cryopreservation liquid and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115968862A true CN115968862A (en) | 2023-04-18 |
Family
ID=85967593
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211678651.7A Pending CN115968862A (en) | 2022-12-26 | 2022-12-26 | Cell cryopreservation liquid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115968862A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116941606A (en) * | 2023-09-19 | 2023-10-27 | 内蒙古原生元生物科技有限公司 | Construction method of cell tissue library of umbilical cord tissue and related products and application thereof |
-
2022
- 2022-12-26 CN CN202211678651.7A patent/CN115968862A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116941606A (en) * | 2023-09-19 | 2023-10-27 | 内蒙古原生元生物科技有限公司 | Construction method of cell tissue library of umbilical cord tissue and related products and application thereof |
| CN116941606B (en) * | 2023-09-19 | 2023-12-05 | 内蒙古原生元生物科技有限公司 | Construction method of cell tissue library of umbilical cord tissue and related products and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110050782B (en) | Stem cell cryopreservation solution and preparation method and cryopreservation method thereof | |
| CN108617638B (en) | Tissue and/or cell cryopreservation protective solution and preparation and application thereof | |
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| US8481253B2 (en) | Cryopreservation of adipose tissue for the isolation of mesenchymal stem cells | |
| EP1976980B1 (en) | Viable cells from frozen umbilical cord tissue | |
| CN106465710B (en) | Adipose tissue cryopreservation liquid and adipose tissue cryopreservation method | |
| CN109090102A (en) | A kind of general serum-free frozen stock solution of mescenchymal stem cell and preparation method thereof | |
| CN106982821A (en) | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof | |
| AU2009228141A1 (en) | Mehtod, system, and apparatus for hypothermic collection, storage, transport and banking of birth tissue | |
| CN110079498B (en) | Human placenta mesenchymal stem cell and preparation method and application thereof | |
| CN104560869B (en) | A kind of method for preparing chorion mescenchymal stem cell | |
| CN109511651A (en) | A kind of preparation method of human umbilical cord mesenchymal stem cells serum-free frozen stock solution | |
| JP2019131573A (en) | Method for cryopreservation of umbilical cord tissue | |
| CN111602652A (en) | Umbilical cord mesenchymal stem cell cryopreservation protective solution | |
| JPWO2016076428A1 (en) | Method for cryopreserving umbilical cord blood and peripheral blood and solution for cryopreservation | |
| CN115968862A (en) | Cell cryopreservation liquid and application thereof | |
| CN113261557A (en) | Stem cell cryopreservation liquid and stem cell cryopreservation method | |
| CN111838130A (en) | Protective solution and preparation method and application thereof | |
| CN115247147A (en) | Culture and cryopreservation method of umbilical cord mesenchymal stem cells | |
| CN113973805A (en) | Cell cryopreservation kit and using method thereof | |
| CN107711823A (en) | The cells frozen storing liquid and its application that a kind of normal temperature preserves | |
| CN113854280B (en) | Low-temperature preservation solution and preparation method and application thereof | |
| JP2020078282A (en) | Method of preserving synovial membrane tissue and method of separating and culturing stem cells from preserved synovial membrane tissue | |
| CN115152743A (en) | Preparation method of human umbilical cord mesenchymal stem cell serum-free frozen stock solution | |
| CN104938479A (en) | Composition and its use, umbilical cord preservation preparation and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |