CN111607561B - Construction method of large-scale, high-flux and high-safety stem cell library - Google Patents
Construction method of large-scale, high-flux and high-safety stem cell library Download PDFInfo
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Abstract
The application provides a method for constructing a large-scale, high-flux and high-safety stem cell library, which mainly comprises the following steps: s1: filling stem cell suspension into a wheat tube for non-vitrification freezing storage; s2: and placing the wheat tube with the frozen stem cell suspension in a buried constant-temperature metal bath for resuscitating. The method for constructing the large-scale, high-flux and high-safety stem cell library has the following technical effects: according to the application, the straws commonly used in the vitrification cryopreservation method in the field are applied to non-vitrification cryopreservation, and the submerged constant-temperature metal bath resuscitating method is matched, so that resuscitating time and resuscitating uniformity can be remarkably improved, indexes such as survival rate, resuscitating rate and recovery rate of stem cells after resuscitating are greatly improved, a rapid thawing principle of non-vitrification cryopreservation is realized, and activity of the cells after resuscitating is maintained to the maximum extent.
Description
Technical Field
The application belongs to the field of stem cells, and particularly relates to a method for constructing a large-scale, high-flux and high-safety stem cell library.
Background
The stem cell bank is three-level management, namely a primary stem cell bank (seed stem cell bank), a stem cell bank and a working stem cell bank.
Primary stem Cell Bank (PCB): the primary cell is also called a cell seed (cell seed), which refers to a cell population which is obtained by culturing tissues or cells from a single individual in a certain mode and has uniform identification properties. The primary stem cell bank is a collection of containers which are dispensed in a predetermined number and store primary cells under a predetermined condition. The stock cells of the primary stem cell bank can be used for preparing the stem cell bank after being qualified by verification.
Stem Cell Bank (Master Cell Bank, MCB): the cell population with uniform properties, which is obtained by one-time preparation through cell culture of a primary stem cell bank to a specific multiplication level or passage level, is divided into a certain number of containers and stored under certain conditions. The storage cells of the stem cell bank can be used for preparing a working stem cell bank or a cell preparation after being qualified by verification.
Working stem cell bank (Working Cell Bank, WCB): the stem cell library is prepared through culturing the cells to specific multiplication level or passage level, preparing cell population with homogeneous property, packing in certain amount and storing in certain condition. The storage cells of the working stem cell library can be used for preparing cell preparations after being qualified by detection.
In the stem cell bank construction process, after high-quality cell preparation is completed, how to effectively and stably store cells for a long time is particularly important, so that: cell cryopreservation technology is used as a core technology for stem cell bank construction.
At present, the most common technology for freezing and preserving cells is a liquid nitrogen freezing and preserving method, wherein a slow freezing method with a proper amount of protective agent is mainly adopted for freezing and preserving cells, and the key of the cell freezing technology is to reduce the water content in the cells as much as possible and reduce the formation of ice crystals in the cells. The glycerol or dimethyl sulfoxide is used as a protective agent, and the two substances have small molecular weight and high solubility, are easy to penetrate cells, can lower the freezing point, improve the permeability of cell membranes to water, and have no obvious toxicity to the cells. The slow freezing method can make the water in the cells seep out of the cells, and reduce the chance of forming ice crystals in the cells, so that the damage of ice crystals to the cells is reduced, and the common cell freezing storage is a liquid nitrogen storage.
The freezing preservation method can be divided into non-vitrification freezing preservation and vitrification freezing preservation according to whether the freezing protection liquid forms ice crystals after freezing.
The vitrification cryopreservation method is a method of protecting suspension cells by using a glass cryoprotectant formed by combining various high-concentration cryoprotectants, directly putting liquid nitrogen into the suspension cells for cryopreservation, and forming no ice crystals on the cell suspension frozen by the method; the vitrification cryopreservation method has better effect on preserving cell activity, the survival rate can reach more than 90 percent, the vitrification cryopreservation method can be used without complex instruments and equipment, and the vitrification cryopreservation method can be used with liquid nitrogen storage equipment because the concentration of the cryoprotectant in the vitrification cryopreservation liquid is higher, and the vitrification cryopreservation liquid has toxicity to cells at room temperature (but the toxicity is greatly reduced at 4 ℃). Therefore, the whole process of dropping the cryoprotectant must be performed in an ice bath at 4 ℃, in addition, the dropping speed is slow, if the dropping speed is too fast, a very high osmotic pressure is generated outside cells, so that cell membranes are damaged, cell death is caused, so that the cryoprotectant is slowly dropped, the cryoprotectant is allowed to slowly permeate into the cells for a sufficient time to reach the balance inside and outside the cells, at present, the method has been widely applied to the aspect of freezing embryonic stem cells, but is rarely applied to the freezing storage of general cells, and the method may be related to the complex preparation of the cryopreserved liquid and the complex operation before and after the freezing recovery.
The non-vitrification freezing method includes cooling stem cells to-70 deg.c to-80 deg.c in different temperature level in different stages in refrigerator, and then directly adding liquid nitrogen for preservation, or programmed computer cooling and liquid and gas and liquid of liquid nitrogen in certain speed from room temperature to below-100 deg.c and then directly adding liquid nitrogen for preservation.
The most common cell cryopreservation technology is not vitrification, and the currently marketed cryopreservation tube is mainly used for the cell cryopreservation technology of a non-vitrification method; the commercial straws are mainly applied to the cell cryopreservation technology of a vitrification method and are mainly applied to the fields of cryopreservation and resuscitation of germ cells.
Disclosure of Invention
In the traditional stem cell bank construction process, a freezing tube is mainly adopted as a preservation medium of the stem cell bank, freezing and recovery operations mainly depend on manual work, and the large-batch bank construction process has large workload and is easy to make mistakes. In order to solve the technical problems, the application designs and develops a technology for applying the wheat tube non-vitrification cryopreserved cells, and establishes a large-scale, high-flux and high-safety stem cell library construction technology and method.
The specific technical scheme of the application is as follows:
the application provides a method for constructing a large-scale, high-flux and high-safety stem cell library, which mainly comprises the following steps:
s1: filling stem cell suspension into a wheat tube for non-vitrification freezing storage;
s2: and placing the wheat tube with the frozen stem cell suspension in a buried constant-temperature metal bath for resuscitating.
The plugs of wheat Guan Duanbu are two types, are composed of two kinds of polyamine fiber fabrics, are six colors, are convenient to distinguish, are composed of 0.22um hydrophobic films, can only pass through air, cannot pass through liquid and bacteria, and are 0.3ml and 0.5ml in integral of wheat Guan Rong. The method provided by the application is applicable to various stem cells, such as: umbilical cord mesenchymal stem cells, endometrial mesenchymal stem cells, adipose stem cells, placental stem cells, and the like; in the research and development process, when the cryopreserved stem cells are frozen by using the cryopreservation tube, the recovery time is long and uneven, so that the survival rate, recovery rate and the like of the stem cells after recovery are reduced, and the recovery effect is poor. The research finds that when the straws commonly used in the field for vitrification cryopreservation methods are applied to non-vitrification cryopreservation, the recovery efficiency can be remarkably improved by matching with a buried constant-temperature metal bath recovery method, and indexes such as survival rate, recovery rate and recovery rate of stem cells after recovery can be greatly improved.
Further, the step S1 of filling the stem cell suspension into a wheat tube for freezing and preserving comprises the following steps:
s11: automatically filling stem cell suspension into the straws by a straw automatic filling identification system, and sealing, printing and identifying the straws filled with the stem cell suspension;
s12: performing programmed cooling on a wheat tube containing stem cell suspension through a wheat tube programmed cooling system;
s13: the wheat straw frozen storage system is used for containing the wheat straw subjected to program cooling and is placed into a liquid nitrogen tank for freezing storage.
In the traditional stem cell bank construction process, the frozen cells of the frozen tube mainly depend on manual split charging, the uniformity of the manual split charging is poor, the operation condition is limited, and pollution is easy to generate; according to the automatic filling mark system, manual filling is replaced by the automatic filling mark system for the straws, so that manual operation is reduced, and the uniformity of split charging is improved. In the traditional stem cell bank construction process, manually screwing a freezing storage tube cover, sub-packaging 1ml of cell suspension into the freezing storage tube, and screwing the freezing storage tube cover, wherein the overall average speed is 80-120 tubes/hour; the application applies the automatic filling equipment of the straw to aseptically fill the cell suspension, the average filling and coding rate is 240-280 tubes/hour, and the working efficiency is improved. In the traditional stem cell bank construction process, the freezing tube needs to be manually hung and covered, and if the manual cover is not screwed tightly, the risk of cross contamination exists; the heat sealing technology of the automatic filling identification system of the wheat tube is applied to heat seal the two ends of the wheat tube, so that the risk of cross contamination is effectively reduced. In the traditional stem cell bank construction process, the printed ultralow-temperature label is pasted on the wall of the freezing storage tube or the code is manually written, a great amount of manual operation time is needed, and the pasted label is usually easy to fall off; the application applies the automatic straw code spraying equipment, can spray international one-dimensional codes (128 codes) at the middle part of the straw, and the sprayed ink is low-temperature resistant (-196 ℃ condition), and the codes are used as unique identifiers of cell samples, so that the traceability management can be better realized.
Further, in step S2, the straw with the frozen stem cell suspension therein is placed in a buried constant temperature metal bath for resuscitation at 37 ℃.
In the traditional stem cell bank construction process, the freezing storage pipe is placed on a water bath pot or a support (such as a buoy) and placed in the water bath pot for resuscitation, and because the freezing storage pipe is thicker, the resuscitation time is usually 1-3min and the resuscitation is uneven, the application adopts the buried constant temperature metal bath wheat pipe special resuscitator to place the wheat pipe into the special resuscitator for 60 seconds at most, thus realizing the resuscitator process and realizing the even and quick thawing principle of non-vitrification freezing storage.
Further, the freezing or recovering process also comprises safe transportation, wherein the safe transportation is to transport the wheat straws subjected to the program cooling into a freezing storage system for freezing and storing through a dry liquid nitrogen tank, or transport the wheat straws in the freezing storage system into a buried constant-temperature metal bath for recovering through the dry liquid nitrogen tank.
Further, the step S12 of program cooling is to put the program cooling box into a refrigerator with the temperature of minus 80 ℃ for 12 hours.
Further, the automatic straw filling identification system in step S11 includes: the device comprises a control box, a code spraying machine, identification equipment, filling equipment and heat sealing equipment;
the control box is used for controlling the filling equipment to fill the straws, the code spraying machine is used for spraying codes on the straws, the identification equipment is used for identifying bar codes on the straws, and the heat sealing equipment is used for heat sealing the straws Guan Jinhang filled with the stem cell suspension;
the filling equipment comprises a source sample bag, a liquid injection pipe communicated with the source sample bag, a peristaltic pump connected with the liquid injection pipe, and a liquid outlet pipe connected with the bottom end of the liquid injection pipe, wherein the liquid outlet pipe is used for filling stem cell suspension into the straw, the liquid outlet end of the source sample bag is in sealing connection with the end part of the liquid injection pipe, and the source sample bag is provided with a vibration device.
The application improves the traditional automatic filling system of the wheat tube, and improves the original static sample injection device of the open tube into the closed sample injection device which is vertically rotated and uniformly mixed, thereby effectively ensuring the aseptic condition of cells and ensuring the uniform density of the split-packed cell suspension.
Further, the wheat tube program cooling system in step S12 is to put wheat tubes into a wheat tube program cooling box for program cooling, the program cooling box comprises a box body with an opening at the top end and a box cover matched with the box body, the box body is made of EVA foam, a plurality of vertical EVA foam plates are arranged in the box body at equal intervals, a heat conducting core is arranged between two adjacent EVA foam plates, and a plurality of wheat tube slots are arranged on the EVA foam plates and/or the box body.
Further, the number of the heat conducting cores is 3, the number of the EVA foam plates is 2, the thickness of the box body is 10mm, the size of the box body is 120mm long by 70mm wide by 155mm high, the height of the box cover is 20mm, the sizes of the heat conducting cores and the EVA foam plates are 100mm long by 10mm wide by 140mm high, and 24 wheat tube slots are formed in each EVA foam plate and the side walls of the two box bodies parallel to the EVA foam plates.
In the construction process of the traditional stem cell bank, the number of samples processed at one time by the traditional program cooling box/program cooling instrument is less, and is usually 12; the special program cooling box for the wheat tube can be used for containing the wheat tube filled with stem cell suspension, and then the wheat tube is placed in a refrigerator at the temperature of minus 80 ℃ for program cooling, the cooling speed of each wheat tube is more consistent in the cooling process, and compared with the conventional program cooling box for freezing 12 freezing tubes at one time, the special program cooling box for the wheat tube can freeze 96 wheat tubes at one time.
Further, in the step S13, the wheat tube freezing and storing system is characterized in that wheat tubes are firstly placed in a freezing and storing box, then the freezing and storing box is placed in a freezing and storing frame, and then the freezing and storing frame is placed in a liquid nitrogen tank for freezing and storing, the special freezing and storing box for the wheat tubes comprises a shell and a cover matched with the shell, a reticular isolation groove is arranged in the shell, plastic columnar bodies are arranged in each isolation groove of the reticular isolation groove, and a plurality of wheat tube freezing and storing grooves are arranged on each plastic columnar body; the special freezing frame for the wheat tube comprises a frame with an opening on the side face, a plurality of partition plates are arranged on the frame from bottom to top at equal intervals, the length and the width of each partition plate are larger than those of the freezing box, and the distance between every two adjacent partition plates is larger than the height of the freezing box.
Further, a handle is arranged at the top end of the frame.
Each conventional freezing box can be placed with 9*9 =81, and each special freezing box for the wheat straw can be placed with 9×12=1296, and the number of stored samples is 16 times more than that of the conventional freezing box matched with the freezing tube, so that the freezing box can be independently placed in a mobile dry liquid nitrogen tank for convenient transportation, or the freezing box can be placed in a special freezing frame for the wheat straw, and then the freezing frame is placed in the liquid nitrogen tank for convenient taking and placing; each conventional freezing frame can be used for placing 81 x 10=810 freezing pipes, each special freezing frame for wheat pipes can be used for placing 3 special freezing boxes for wheat pipes, namely 1296 x 3=3888 wheat pipes; compared with the traditional freezing frame, the freezing frame provided by the application can store more stored samples by 5 times
Further, two opposite outer side walls of the shell are horizontally provided with through grooves, the left side wall and the right side wall of the frame are respectively provided with a convex block matched with the through grooves, an extrusion plate is arranged above the partition plate, and a spring is vertically arranged between the extrusion plate and the partition plate.
The method for constructing the large-scale, high-flux and high-safety stem cell library has the following technical effects: according to the application, the straws commonly used in the vitrification cryopreservation method in the field are applied to non-vitrification cryopreservation, and the buried constant-temperature metal bath resuscitating method is matched, so that resuscitating time and resuscitating uniformity can be remarkably improved, indexes such as the adherence rate, resuscitating rate and recovery rate of stem cells after resuscitating are greatly improved, a rapid thawing principle of non-vitrification cryopreservation is realized, and cell resuscitating activity is maintained to the maximum extent.
Drawings
FIG. 1 is a flow chart of stem cell cryopreservation;
FIG. 2 is a flow chart of stem cell resuscitation;
FIG. 3 is a schematic structural view of the filling apparatus of embodiment 1;
FIG. 4 is a schematic diagram of a temperature reduction box according to embodiment 2;
fig. 5 is a schematic structural view of a special freezing box for straws in embodiment 3;
FIG. 6 is a schematic view of a part of the structure of the special freezing box for wheat straw in embodiment 3;
fig. 7 is a schematic structural diagram of a special freezing rack for straws in embodiment 3;
fig. 8 is a schematic structural view of a special freezing box for straws in embodiment 4;
fig. 9 is a schematic structural view of a special freezing shelf for wheat straw in embodiment 4;
fig. 10 is a sectional view of the separator and the pressing plate in example 4.
Detailed Description
Example 1
The embodiment provides a filling device, as shown in fig. 3, the filling device includes a source sample bag 1, a liquid injection tube 2 communicated with the source sample bag 1, a peristaltic pump 3 connected with the liquid injection tube 2, a liquid outlet tube 4 connected with the bottom end of the liquid injection tube 2, the liquid outlet tube 4 is used for filling stem cell suspension into a straw, the liquid outlet end of the source sample bag 1 is in sealing connection with the end part of the liquid injection tube 2, and a vibration device 5 is arranged on the source sample bag 1.
Embodiment 1 through setting up the liquid end of source sample bag and annotate the tip sealing connection of liquid pipe, effectually guaranteed the aseptic condition of cell, guaranteed the density homogeneity of the cell suspension of partial shipment simultaneously through setting up vibrator.
Example 2
The embodiment provides a program cooling box, as shown in fig. 4, the program cooling box comprises a box body 6 with an opening at the top end and a box cover 7 matched with the box body 6, wherein the box body 6 is made of EVA foam, a plurality of vertical EVA foam plates 8 are arranged in the box body 6 at equal intervals, a heat conducting core 9 is arranged between two adjacent EVA foam plates 8, and a plurality of wheat tube slots 10 are arranged on the EVA foam plates 8 and/or the box body 6; the quantity of heat conduction core 9 is 3, and the quantity of EVA foam board 8 is 2, and the thickness of box body is 10mm, and the size of box body is long 120mm wide 70mm high 155mm, and the height of lid is 20mm, and the size of heat conduction core 9 and EVA foam board 8 is long 100mm wide 10mm high 140mm, all is equipped with 24 wheat pipe slots 10 on the lateral wall of every EVA foam board 8 and two box body 6 that are parallel with EVA foam board 8.
The box body is matched with the box cover and can be connected through a rotating shaft, the embodiment is not particularly limited, the box body is made of EVA foam materials, the EVA foam plates and the box body are integrally formed, and the heat conducting core is made of alloy materials; the program cooling box special for the wheat tube can be used for containing the wheat tube filled with the stem cell suspension, and then the wheat tube is placed in a refrigerator at the temperature of minus 80 ℃ for program cooling, the cooling speed of each wheat tube is more consistent in the cooling process, and compared with the conventional program cooling box for freezing 12 freezing tubes at one time, the program cooling box special for the wheat tube can freeze 96 wheat tubes at one time.
Example 3
The embodiment provides a freezing box and a freezing frame, as shown in fig. 5 and 6, the freezing box special for wheat tubes comprises a shell 11 and a cover matched with the shell 11, a net-shaped isolation groove is arranged in the shell 11, a plastic column body 12 is arranged in each isolation groove 13 of the net-shaped isolation groove, and a plurality of wheat tube freezing grooves 14 are arranged on each plastic column body 12; as shown in fig. 7, the special freezing frame for wheat tube comprises a frame 15 with an opening on the side, a plurality of partition boards 16 are arranged at equal intervals from bottom to top on the frame, the length and width of each partition board 16 are larger than those of the freezing box, the distance between two adjacent partition boards 16 is larger than the height of the freezing box, and a handle is arranged at the top end of the frame 15.
The cover and the shell are matched in a sleeving manner, a shaft connection manner, a clamping manner and the like, the shell is a box with an opening at the top end, the box is made of PVC materials, the special freezing box for the wheat tube can be used for freezing a large quantity of wheat tubes, and the freezing frame is made of stainless steel materials; each conventional freezing box can be placed with 9*9 =81, and each special freezing box for the wheat straw can be placed with 9×12=1296, and the number of stored samples is 16 times more than that of the conventional freezing box matched with the freezing tube, so that the freezing box can be independently placed in a mobile dry liquid nitrogen tank for convenient transportation, or the freezing box can be placed in a special freezing frame for the wheat straw, and then the freezing frame is placed in the liquid nitrogen tank for convenient taking and placing; each conventional freezing frame can be used for placing 81 x 10=810 freezing pipes, each special freezing frame for wheat pipes can be used for placing 3 special freezing boxes for wheat pipes, namely 1296 x 3=3888 wheat pipes; the freezing storage rack provided by the application can store more stored samples by 5 times than the traditional freezing storage rack.
Example 4
The embodiment provides a freezing box and a freezing frame, which are different from embodiment 3 in that, as shown in fig. 8 and 9, two opposite outer side walls of a casing 11 are horizontally provided with through grooves 17, left side walls and right side walls of a frame 15 are respectively provided with a protruding block 18 matched with the through grooves 17, a pressing plate 19 is arranged above a partition plate 16, and a spring 20 is vertically arranged between the pressing plate 19 and the partition plate 16 as shown in fig. 10.
The protruding blocks can be integrally formed with the frame, the springs, the extrusion plates and the partition plates are fixedly connected, the sum of the thickness of the extrusion plates, the length of the compressed springs and the height of the freezing storage box is smaller than the distance between two adjacent partition plates, the cross sections of the through grooves and the protruding blocks can be semicircular in the figure 1, square or rectangular and the like, and the application is not limited in particular; when the special freezing box for the wheat tube is used, two protruding blocks of the corresponding frames of the two through grooves on the shell of the special freezing box for the wheat tube are inserted between the two adjacent partition boards, the extruding plate is pressed downwards at the moment, the extruding plate moves downwards to compress the spring, after the special freezing box for the wheat tube is fully placed between the two partition boards, the hand is loosened, the special freezing box for the wheat tube is firmly extruded between the two partition boards by the upward elasticity of the compressing spring, and the phenomenon that the special freezing box for the wheat tube in the special freezing frame for the wheat tube is stressed to move when being in a liquid nitrogen tank is avoided.
Example 5
The embodiment provides a method for constructing a large-scale, high-flux and high-safety stem cell library, which mainly comprises the following steps as shown in fig. 1:
s11: automatically filling stem cell suspension into the straws by a straw automatic filling identification system, and sealing, printing and identifying the straws filled with the stem cell suspension;
s12: placing wheat straw into the wheat straw program cooling box provided in the embodiment 2, and then placing the program cooling box into a refrigerator with the temperature of minus 80 ℃ for 12 hours to carry out program cooling;
s13: firstly, placing a wheat tube into a freezing box of the embodiment 3, then placing the freezing box of the embodiment 3 into a freezing frame of the embodiment 3, and then placing the freezing frame of the embodiment 3 into a liquid nitrogen tank for freezing;
the freezing and storing process also comprises safe transportation, wherein the safe transportation is to transport the wheat straw subjected to the program cooling into a freezing and storing system through a dry liquid nitrogen tank for freezing and storing;
step S11, the automatic straw filling identification system comprises the following steps: control box, ink jet numbering machine, identification equipment, filling equipment and heat sealing equipment that embodiment 1 provided;
the control box is used for controlling the filling equipment to fill the straws, the code spraying machine is used for spraying codes on the straws, the identification equipment is used for identifying bar codes on the straws, and the heat sealing equipment is used for heat sealing the straws Guan Jinhang filled with the stem cell suspension;
s2: placing the frozen stem cell suspension in a buried constant-temperature metal bath, and regulating the temperature to 37 ℃ for resuscitating;
as shown in fig. 2, the temperature of the buried metal bath is adjusted to 37 ℃ first, whether the temperature is accurately adjusted is judged, if not, the temperature is readjusted, if so, the wheat tube is taken out from the liquid nitrogen tank, put into the preheated buried metal bath, the cell melting state is judged, if melted, the sealing head is aseptically subtracted to obtain a cell suspension, and if not melted, the wheat tube is continuously put into the preheated buried metal bath for resuscitation until melted.
Test example 1
Preparation 1.0 x 10 6 Each ml of umbilical mesenchymal stem cell suspension (22 ml with 2ml allowance) was averaged 3 times for cell count and viability assay (trypan blue exclusion method), and 10% DMSO was added for use.
The umbilical mesenchymal stem cell suspension is filled into 20 straws according to the method of the embodiment 5, the umbilical mesenchymal stem cell suspension is filled into 20 freezing tubes of a control group, the umbilical mesenchymal stem cell suspension is respectively sealed and then is frozen for 12 hours at the temperature of-80 ℃ in a pre-cooling program cooling box at the temperature of 4 ℃, the umbilical mesenchymal stem cell suspension is transferred into the freezing box and then is frozen at the same height position in a liquid nitrogen tank, the conventional freezing tube, the program cooling box and the freezing box are used for the control group, the freezing tube is a Corning freezing tube, the program cooling box is a Corning Coolcell LX program cooling box, the program cooling box is a product number 432001, and the freezing box is CryoKING with the model number of 98-1213.
After one week, the straws of the test group and the freezing tubes of the control group, which are filled with the umbilical cord mesenchymal stem cell suspension, are taken out from the liquid nitrogen, the freezing tubes are put into a water bath kettle at 37 ℃ for resuscitation for 120s, the straws are put into a buried metal bath at 37 ℃ for resuscitation for 60s, and the umbilical cord mesenchymal stem cell suspension after each tube is resuscitated is counted and the activity rate is measured.
Cell recovery was calculated: recovery of umbilical cord mesenchymal stem = total umbilical cord mesenchymal stem cells after resuscitation/total umbilical cord mesenchymal stem cells before resuscitation.
Calculating the cell recovery rate: umbilical cord mesenchymal stem cell viability after recovery/umbilical cord mesenchymal stem cell viability before recovery;
the test results are shown in Table 1.
TABLE 1 comparison of cell viability before and after thawing for two groups of resuscitation times
Analysis of results
The analysis of the experimental results can be obtained: the cell activity rate after the straw group is recovered is obviously higher than that of the cryopreserved straw group (P < 0.05); the recovery rate of the straw group and the cryopreserved straw group after resuscitation is not obviously different (P > 0.05); the recovery rate after the straw group recovery is significantly higher than that of the cryopreserved straw group (P < 0.05).
In summary, the present application is not limited to the preferred embodiments, but is intended to cover modifications and equivalent arrangements included within the scope of the appended claims and their equivalents.
Claims (3)
1. The method for constructing the large-scale, high-flux and high-safety stem cell library is characterized by mainly comprising the following steps of:
s1: filling stem cell suspension into a wheat tube for non-vitrification freezing storage;
s11: automatically filling stem cell suspension into the wheat tube through a wheat tube automatic filling identification system, and sealing, printing and identifying the wheat tube filled with the stem cell suspension; the automatic straw filling identification system comprises: the device comprises a control box, a code spraying machine, identification equipment, filling equipment and heat sealing equipment; the control box is used for controlling the filling equipment to fill straws, the code spraying machine is used for spraying codes on the straws, the identification equipment is used for identifying bar codes on the straws, and the heat sealing equipment is used for heat sealing of the straws Guan Jinhang filled with stem cell suspensions; the filling device further comprises: the device comprises a source sample bag (1), a liquid injection pipe (2) communicated with the source sample bag (1), a peristaltic pump (3) connected with the liquid injection pipe (2) and a liquid outlet pipe (4) connected with the bottom end of the liquid injection pipe (2), wherein the liquid outlet pipe (4) is used for filling stem cell suspension into a wheat pipe, the liquid outlet end of the source sample bag (1) is in sealing connection with the end part of the liquid injection pipe (2), and a vibration device (5) is arranged on the source sample bag (1);
s12: performing programmed cooling on a wheat tube containing stem cell suspension through a wheat tube programmed cooling system; the program cooling is to put the program cooling box into a refrigerator with the temperature of minus 80 ℃ and keep for 12 hours;
the program cooling box comprises a box body (6) with an opening at the top end and a box cover (7) matched with the box body (6), wherein the box body (6) is made of EVA foam, a plurality of vertical EVA foam plates (8) are arranged in the box body (6) at equal intervals, a heat conducting core (9) is arranged between every two adjacent EVA foam plates (8), and a plurality of straw slots (10) are formed in the EVA foam plates (8) and/or the box body (6);
s13: the wheat straw frozen storage system is used for containing the wheat straw subjected to the program cooling and is placed into a liquid nitrogen tank for freezing; the freezing and storing process also comprises safe transportation, wherein the safe transportation is to transport the wheat straw subjected to the program cooling into a freezing and storing system through a dry liquid nitrogen tank for freezing and storing;
the wheat tube freezing and storing system comprises a wheat tube, a freezing and storing box, a freezing and storing frame and a liquid nitrogen tank, wherein the wheat tube is firstly placed in the freezing and storing box, the freezing and storing box is placed in the freezing and storing frame, the freezing and storing frame is placed in the liquid nitrogen tank for freezing and storing, the special freezing and storing box for the wheat tube comprises a shell (11) and a cover matched with the shell (11), a reticular isolation groove (13) is arranged in the shell (11), a plastic column body (12) is arranged in each isolation groove (13) of the reticular isolation groove, and a plurality of wheat tube freezing and storing grooves (14) are arranged on each plastic column body (12); the special freezing frame for the wheat tube comprises a frame (15) with an opening on the side surface, wherein the frame (15) is provided with a plurality of partition boards (16) at equal intervals from bottom to top, the length and the width of each partition board (16) are larger than those of the freezing box, and the distance between two adjacent partition boards (16) is larger than the height of the freezing box; two opposite outer side walls of the shell (11) are horizontally provided with through grooves (17), the left side wall and the right side wall of the frame (15) are respectively provided with a convex block (18) matched with the through grooves (17), an extrusion plate (19) is arranged above the partition plate (16), and a spring (20) is vertically arranged between the extrusion plate (19) and the partition plate (16);
s2: placing the wheat tube with the frozen stem cell suspension in a buried constant-temperature metal bath for resuscitation, wherein the resuscitation is performed at 37 ℃.
2. The method for constructing a large-scale, high-throughput and high-safety stem cell bank according to claim 1, wherein the recovery process further comprises safe transportation, wherein the safe transportation is to transport the straws in the freezing storage system into a buried constant-temperature metal bath for recovery by a dry liquid nitrogen tank.
3. The method for constructing a large-scale, high-throughput and high-safety stem cell bank according to claim 1, wherein the number of the heat conducting cores (9) is 3, the number of the EVA foam sheets (8) is 2, the thickness of the box body is 10mm, the size of the box body is 120mm long by 70mm wide by 155mm high, the height of the box cover is 20mm, the sizes of the heat conducting cores (9) and the EVA foam sheets (8) are 100mm long by 10mm wide by 140mm high, and 24 straw slots (10) are arranged on each of the EVA foam sheets (8) and the side walls of the box body (6) parallel to the EVA foam sheets (8).
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