CN104336005A - Blood bag gradual freezing storage device and method of use thereof - Google Patents

Abstract

The invention relates to a blood bag gradually freezing and storing device and a using method thereof, wherein the blood bag gradually freezing and storing device comprises: a main body shell, more than one layer of blood bag placing space and more than two Teflon freezing bags; the main body shell is provided with an upper cover which can be opened and closed; the blood bag placing space is used for containing a blood bag to be cryopreserved; the teflon gradually-freezing bag is in contact with a blood bag to be frozen, and the teflon gradually-freezing bag is filled with a gradually-freezing agent. When the freezing device is assembled, the blood bag to be frozen and the freezing bag are arranged in the main body of the blood bag freezing storage device in a laminating mode, and the Teflon freezing bag is in direct contact with the blood bag to assist the blood bag to achieve the desired effect of slowly cooling in the freezing process.

Description

Blood bag gradual freezing storage device and method of use thereof

technical field

The invention relates to a blood bag freezing storage device, in particular to a blood bag gradual freezing storage device and a use method thereof.

Background technique

When storing cells by freezing, it is necessary to go through a slow cooling process to avoid rapid cooling or unstable cooling that will cause cell death due to crystalline condensation of water in the cells during the freezing process. Successful freezing of cells requires two basic conditions, one is to add a cryoprotectant, and the other is to cool down at an optimal cooling rate. The commonly used cryoprotectant is dimethyl sulfoxide (DMSO). DMSO is a very small molecule that can enter the cell through the cell membrane to slow down the size and rate of ice crystals formed by water in the cell and protect the cell during frozen storage and thawing. The process is not damaged, increasing the cell survival rate. However, when the concentration of DMSO is high, it is also cytotoxic, so it should be controlled below 10% (wt) during cryopreservation, and the best cooling rate should be combined to achieve the best cryopreservation of cells.

In order to achieve the best cooling rate in commercially available cryo boxes, 100% isopropyl alcohol (isopropyl alcohol) is filled between the inner box storage layer and the outer shell as a gradual freezing agent, which can drop the temperature every minute. 1 ℃ temperature to stabilize the cooling, the lowest can reach -80 ℃, and can maintain low temperature for a long time. Isopropanol can help achieve slow cooling in the process of freezing cells. Using this substance, the cooling rate can reach approximately -1°C/min, and the effect of smooth cooling curve can also be obtained in the freezer. However, isopropanol is highly volatile and easily absorbs moisture in the air, so after a long period of use, its concentration will gradually decrease and affect its temperature control performance. Therefore, the cryotube gradual freezing box produced by Kelowna International Technology Co., Ltd. does not need to add isopropanol to achieve the gradual freezing effect, and because it can quickly return to temperature, it can shorten the time for re-use. But so far, known commercially available gradual freezing boxes (such as Mr. Frosty produced by Nalgene) are only for cryopreservation of cryotubes, and there is no public use of related products such as blood bag gradual freezing boxes for blood bag freezing.

The American Association for the Accreditation of Cell Therapy (FACT) specifies that umbilical cord blood must be stored in antifreeze blood bags, because antifreeze blood bags are highly airtight and can reduce the chance of contamination. The preservation method generally adopted in the industry at present is to pack the collected umbilical cord blood into, for example, a cryo preserved bag (cryo preserved bag) produced by Pall Company (USA). The Pall freezer bag is a new type of blood bag that consists of two divided parts, one with a capacity of 20ml and the other with a capacity of 5ml. The latter can be used for self-expansion before stem cell transplantation, and then transplanted to patients together with the former, or used to treat damaged tissues and organs. In order to prevent cross-contamination, an additional layer of bags will be added to the outside of the freezer bag and then put into an aluminum alloy box, and then the metal box will be placed in a computerized temperature-reducing system for program freezing. The cooling rate is - 1°C/min. When the temperature drops to -80°C, the umbilical cord blood will be transferred to a liquid nitrogen box and stored in a gaseous state (keep a low temperature of ≤ -190°C) for a long time (more than one year). However, the controllable rate cooling instrument is expensive. In addition to greatly increasing the cost of stem cell storage and transplantation, a drop of 1°C per minute is the standard value for a small amount of cryopreserved cells in cryovials. It can be used as a method for storing a large number of cells in blood bags. Reference value, but the optimal cooling rate is slightly adjusted due to the number of cells, so the survival rate of cells after thawing in a blood bag is lower than that in a frozen tube. At present, the laws and regulations of various countries in the world stipulate that the survival rate of cells after thawing in a blood bag is as high as More than 70% is one of the release standards for clinical use.

Contents of the invention

In view of this, the purpose of the present invention is to provide a blood bag gradual freezing storage device and its use method.

In order to achieve the above purpose, the present invention provides a blood bag gradual freezing storage device, comprising:

A main body shell, which is provided with an openable and airtight upper cover, which is sealed after the blood bag gradual freezing storage device is assembled;

More than one layer of blood bag insertion space, the blood bag insertion space accommodates blood bags to be frozen; and

More than two Teflon gradual freezing bags, the Teflon gradual freezing bags are in contact with the blood bags to be frozen, and the Teflon gradual freezing bags are filled with materials that can reduce the temperature to -1°C/min gradual freezing agent.

As a preferred solution of the aforementioned blood bag gradual freezing storage device, the gradual freezing agent is isopropanol. The size of the gradual freezing bag is based on the length and width of the inner edge of the main body, and the ratio of the weight of the gradual freezing agent filled (g) to the area of the gradual freezing bag (cm ² ) is 0.7~0.9. The best carrying capacity of the bag.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, wherein the blood bag insertion space and the Teflon gradual freezing bag are alternately stacked and arranged at the bottom of the blood bag gradual freezing storage device inside the body.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, the upper and lower layers of the blood bag insertion space are connected to the Teflon gradual freezing bag, and the upper and lower layers of the inserted blood bag can be were exposed to the Teflon gradual freezing bags.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, wherein the upper cover of the main body shell is tightly fitted to the main body of the blood bag gradual freezing storage device in a press-fit manner, so as to avoid contamination of the contents and facilitate transportation and storage. stack.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, wherein the inner peripheral edge of the upper cover is provided with an annular groove that is closely connected with the opening peripheral edge of the main body shell.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, wherein the upper cover of the main body casing is tightly fitted to the main body of the blood bag gradual freezing storage device in a buckled manner, so as to avoid contamination of the contents and facilitate transportation and storage. stack.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, the main body shell and the upper cover are respectively provided with more than one set of interlocking external buckle devices.

As a preferred solution of the aforementioned blood bag gradual freezing storage device, the main body casing and the upper cover are made of freeze-resistant plastic, paper or metal.

As a preferred solution of the aforementioned blood bag gradual freezing storage device, wherein the main body shell and the upper cover are made of PP plastic.

As a preferred solution of the above-mentioned blood bag gradual freezing storage device, the blood bag insertion space is a metal box that can be disassembled separately, or a partition that can be erected on the protruding point of the inner wall of the main body shell. The space formed, the blood bag insertion space must have a height that enables the upper and lower outer surfaces of the inserted blood bag to be in contact with the Teflon gradual freezing bag.

As a preferred solution of the above-mentioned gradual freezing storage device for blood bags, the size of the gradual freezing bag is based on the principle of matching the length and width of the inner edge of the main body shell, and the weight (g) of the gradual freezing agent filled therein/the area of the gradual freezing bag (cm ² ) The ratio is 0.7~0.9, which takes into account the better carrying capacity of the gradual freezing bag used.

The "blood bag" referred to in the present invention is broadly defined as a frozen bag containing cell culture therein. As used herein, the cell culture can be a cell culture that has been cultured in large quantities in vitro, including (but not limited to) stem cell culture, or umbilical cord blood collected from a living body.

The present invention also provides a method for using a blood bag gradual freezing storage device, which includes the following steps:

Collect cell culture or cord blood in freezer bags;

Put one of the Teflon gradual freezing bags filled with gradual freezing agent into the bottom of the main body of the blood bag gradual freezing preservation device;

Set up a layer of blood bags on the top of the Teflon gradual freezing bag to put into the space;

placing a frozen bag containing cell culture or cord blood therein into said blood bag insertion space;

One of the Teflon gradual freezing bags filled with gradual freezing agent is placed above the blood bag insertion space;

Repeat the overlapping insertion steps of the blood bag and the Teflon gradual freezing bag;

Sealing the assembled blood bag gradual freezing storage device and placing it at -80°C for 6-10 hours to gradually cool down; and

The blood bag gradual freezing storage device whose temperature has dropped to -80° C. was moved into liquid nitrogen for storage.

As a preferred solution of the above-mentioned method of using the blood bag gradual freezing storage device, the temperature drop rate of the blood bag in the blood bag gradual freezing storage device is 1° C. per minute.

As a preferred solution of the above-mentioned method for using the blood bag gradual freezing storage device, the cell culture is a stem cell culture.

The blood bag gradual freezing storage device provided by the present invention, when the gradual freezing device is assembled, the blood bag to be frozen and the gradual freezing bag are stacked and placed inside the main body of the blood bag gradual freezing storage device, and The Teflon gradual freezing bag is in direct contact with the blood bag, assisting the blood bag to achieve the desired slow cooling effect during the freezing process; it has the function of gradual freezing, and the cost is low; and the cell culture is placed in the blood bag for gradual freezing preservation In the device, the survival rate of a large number of cells stored in blood bags after thawing can be improved.

Description of drawings

Fig. 1 is the perspective view of the embodiment main body of a blood bag gradual freezing storage device according to the present invention;

Fig. 2 is a cross-sectional view of an embodiment of a blood bag gradual freezing preservation device assembled by a sandwich method of the present invention;

Figure 3 is the standard ideal cooling curve of the cell freezing process (data source: Transfus Med Rev. 1997 Jul; 11(3):224-33);

Fig. 4A is the temperature drop curve measured at different sampling points including two layers of empty iron clips that have not been placed in the blood bag during the cryopreservation process of the blood bag gradual freezing preservation device arranged and assembled in a sandwich manner according to the present invention;

Fig. 4B is the temperature measured at different sampling points of the blood bag gradual freezing preservation device arranged and assembled in a sandwich manner in the process of cryopreservation, including 2 layers of gradual freezing boxes (CryoBag) that have been placed into blood bags cooling curve;

Fig. 5 is the temperature drop curve measured at different sampling points of the blood bag gradual freezing storage device arranged and assembled in other ways during the cryopreservation process, including two layers of the gradual freezing box (CryoBag) that has been placed in the blood bag;

Fig. 6A is a graph of the cell survival rate measured after thawing of mesenchymal stem cells (MSCs) frozen by the blood bag gradual freezing preservation method of the present invention;

Fig. 6B is a diagram of the appearance and growth characteristics of MSCs observed under a microscope after cryopreservation of mesenchymal stem cells (MSCs) by the blood bag gradual freezing preservation method of the present invention for 3 days after recovery;

Fig. 7A is the first batch of MSCs cryopreserved by the blood bag gradual freezing preservation method of the present invention, after thawing and induction of differentiation, they all have the ability to differentiate into osteoblasts, chondrocytes and adipocytes;

Fig. 7B is the second batch of MSCs cryopreserved by the blood bag gradual freezing preservation method of the present invention, after thawing and induced differentiation, all of them have the ability to differentiate into osteoblasts, chondrocytes and adipocytes;

Fig. 7C shows the third batch of MSCs cryopreserved by the blood bag gradual freezing preservation method of the present invention. After thawing and induction of differentiation, all MSCs have the ability to differentiate into osteoblasts, chondrocytes and adipocytes.

[Description of main component symbols]

1- blood bag gradual freezing storage device; 10- main body shell; 12- upper cover;

20-Blood bag placement space;

30-gradually freezing bag;

40 - cryopreservation bag.

Detailed ways

Other characteristics and advantages of the present invention will be further exemplified and described in the following examples, and these examples are only for auxiliary illustration, and are not intended to limit the scope of the present invention.

Referring first to Fig. 1 and Fig. 2, an embodiment of the structure and assembly method of a blood bag gradual freezing storage device 1 of the present invention will be described in detail. As shown in Figure 1, the blood bag gradual freezing storage device 1 of the present invention includes a main body shell 10, more than one layer of blood bag insertion space 20 and more than two Teflon (Teflon) gradual freezing bags 30, the The main body casing is provided with an upper cover 12 that can be opened and sealed. Each layer of blood bag insertion space 20 can be used to place more than two blood bags. The Teflon (Teflon) gradual freezing bag 30 is filled with gradually The cryogen is respectively installed on the upper and lower parts of each layer of blood bag insertion space 20 during assembly, and is in direct contact with the blood bag.

In order to achieve the best cryopreservation effect, the internal capacity of the main body shell 10 is based on the principle that it can accommodate at most 2 layers of blood bags and 3 gradually freezing bags 30, that is, the single blood bag of the present invention is gradually frozen and stored As far as the device 1 is concerned, the upper limit of the load is preferably 4 blood bags, and at most 2 blood bags can be placed in the single-layer blood bag insertion space 20, and the size of the gradual freezing bag 30 can just match the length of the inner edge of the main body. , wide is better. The inside of Teflon gradual freezing bag 30 is filled with gradual freezing agent (a known preferred gradual freezing agent is 100% isopropyl alcohol (isopropyl alcohol)), wherein the ratio of the filled gradual freezing amount to the area of the gradual freezing bag 30 is, weight (g)/area of gradual freezing bag (cm ² ) = 0.7~0.9. For example, for a gradual freezing bag 30 whose inner edge is 21 cm x 13.5 cm in length and width, it can preferably be filled with 200 grams of 100% isopropanol.

An example of the assembly method of the blood bag gradual freezing storage device 1 of the present invention: The gradual freezing bag 30 is pre-cooled in a -30°C refrigerator for more than 2.5 hours. Put the collected single blood bag into an iron clip (this combination is hereinafter referred to as the cryopreservation bag 40), and temporarily store it in a refrigerator at 4°C. After all the cryopreservation bags 40 are prepared, the blood of the present invention Assembly of bag gradual freezing storage device 1. The assembly method is preferably alternately stacked and arranged in a sandwich method (as shown in Figure 2), wherein the gradual freezing bag 30 of the present invention is layered on the top and bottom of the freezer bag 40, so that the freezer bag 40 is arranged Between the upper and lower progressively freezing bags 30 , the upper and lower sides of the cryopreservation bag 40 can directly contact the gradually freezing bags 30 . A single-layer freezer bag 40 can be placed horizontally between the gradual freezing bags 30, and there are at most two freezer bags 40 in a single layer, and it is confirmed that the freezer bags 40 are evenly stressed on the gradual freezing bag 30, and the gradual freezing bag 30 and the gradual freezing bag are completed. After the cryopreservation bags 40 are stacked, the upper cover 12 of the body shell 10 can be covered. The upper cover 12 and the gradual freezing device body 10 can be tightly closed by pressing or buckling. After the assembly is completed, put the gradual freezing storage device 1 in a -80°C refrigerator for more than 6 hours, preferably 6-10 hours, and gradually cool it down to -80°C before moving it into a liquid nitrogen tank for storage.

Cell freezing must go through a slow cooling process, so that the water in the cells will form vitrified solidification, instead of rapidly condensing into irregular crystals and damaging the cells. The optimal rate of cooling to crystal formation (Pre-freeze) is 1°C per minute (-1°C/min). Its standard ideal cooling curve (STD) is shown in Figure 3 (excerpted from Transfus Med Rev. 1997 Jul; 11(3):224-33).

Therefore, the temperature drop curve obtained during the cooling process of the gradual freezing device assembled in the sandwich stacking method (hereinafter referred to as the sandwich freezing method) and other arrangements of the present invention is further tested to understand the blood bag gradual freezing storage device 1 of the present invention. cell freezing effect. During the sandwich cryopreservation process, the measured cooling curves of the two layers of empty iron clips that had not been placed in the blood bag were consistent, and the cooling rate was close to the STD of 1 °C per minute (as shown in Figure 4A). In the blood bag gradual freezing preservation device 1 assembled by the sandwich method of the present invention, the two-layer cryopreservation bag 40 has a short exothermic time and a small exothermic temperature difference (ΔT) (1.6-2.6°C) during the entire cryopreservation process. It is slightly slower than STD, but it has a good effect on storing a large number of cells in blood bags, which shows that the cooling conditions of the two-layer cryopreservation bags 40 are similar, the cooling is slow, and the exothermic temperature difference is small (see Figure 4B). And directly stack with two-layer cryopreservation bags 40, then place the cryopreservation method (as shown in Figure 5) of gradually freezing bags on the upper and lower layers of the two-layer cryopreservation bags 40, it can be found from its cooling curve that the two cryopreservation The exothermic time of the liquid crystal in the bag 40 is long and the exothermic temperature difference (ΔT) is large, which means that the gradual freezing effect of the sandwich freezing method in the present invention is better than that of the direct stacking blood bag freezing method.

Comparison of cell viability (%). Use other cryopreservation methods such as blood bag direct stack cryopreservation method and sandwich cryopreservation method to gradually freeze the cells at -80°C, and then store them in liquid nitrogen. After taking out and thawing, count them in the cell cryopreservation solution cell. The results showed that MSCs (mesenchymal stem cells) thawed after cryopreservation by the sandwich method had a better cell survival rate (90.89%), which was significantly different from that of the blood bag direct stack cryopreservation method (Figure 6A). As for the observation results of cell morphology (as shown in FIG. 6B ), MSCs were cultured on plates for 3 days after thawing, and it was found that these cells still had the characteristics of sticking growth after recovery.

The MSCs that are gradually frozen using the gradual freezing method of the present invention are thawed after freezing, and then the cell surface antigen identification and in vitro differentiation ability analysis are carried out. After staining the cells with a fluorescent specific antibody, the flow cytometer is used to detect whether the cells express MSC-specific markers.

Table 1.

The results in Figure 7A-Figure 7C and Table 1 show that the proportion of cells expressing CD105, CD90, and CD73 measured after thawing of the 3 batches of MSC cells that were gradually frozen was greater than 95%, and the proportion of cells that did not express Negative Cocktail The cell population of markers (CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR) is greater than 98% (that is, the expression of negative markers <2%), and the results are all in line with the definition of human by the international Society for cellular therapy (ISCT) Antigen expression of mesenchymal stem cells (MSC) (please refer to Cytotherapy (2006) Vol. 8, No. 4, 315-317 for the definition of MSC according to ISCT).

After the cells are attached to the plate, they are guided to differentiate into "osteogenic", "cartilage" and "fat" cells, and the differentiation of the cells is identified by staining. The staining methods and color development results of various cells are listed in Table 2 below.

Table 2.

The results in Figure 7A-Figure 7C and Table 2 show that 3 batches (SW01, SW02, SW03) of gradually frozen MSCs have the ability to differentiate into osteoblasts, chondrocytes and adipocytes after being induced to differentiate. The results are in line with the differentiation potential of human mesenchymal stem cells (tri-lineage differentiation capacity) defined by ISCT.

Based on the above-mentioned cell measurement results, compared with other blood bag cryopreservation methods, the blood bag gradual freezing preservation device 1 of the present invention and the cryopreservation method using it can provide cooling conditions that are more conducive to cell cryopreservation, and make a large number of cells cryopreservation. A better survival rate (90.89%) was maintained in the blood bag. Moreover, the MSCs frozen and stored by the blood bag gradual freezing storage device 1 of the present invention still have the characteristics and differentiation ability of MSCs after thawing, and all meet the ISCT release standard for clinical use of MSCs.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Therefore, all equivalent modifications or changes made by using the contents of the description and drawings of the present invention should also fall within the scope of patent protection of the present invention.

Claims (10)

1. blood bag gradually freezes a save set, it is characterized in that, comprising:

One main body cover, its be provided with one can for opening, airtight upper cover, and gradually freeze after save set has been assembled in described blood bag and present sealing;

Space inserted by blood bag more than one deck, and described blood bag is inserted space and is equipped with for frozen blood bag; And

The Teflon of more than two gradually freezes bag, and described Teflon gradually freezes bag and contacts with for frozen blood bag, and described Teflon gradually freeze in bag be filled with can make rate of temperature fall reach-1 DEG C/min gradually freeze agent.

2. blood bag according to claim 1 gradually freezes save set, it is characterized in that, described gradually to freeze agent be isopropyl alcohol.

3. blood bag according to claim 1 gradually freezes save set, it is characterized in that, space inserted by described blood bag and described Teflon gradually freezes bag with mutual stacked manner, is arranged in the body interior that described blood bag gradually freezes save set from the bottom to top.

4. blood bag according to claim 3 gradually freezes save set, it is characterized in that, the upper and lower layer that space inserted by described blood bag all gradually freezes bag with described Teflon and is connected, and the above and below of the blood bag of inserting can touch described Teflon respectively gradually freezes bag.

5. blood bag according to claim 1 gradually freezes save set, it is characterized in that, the main body that the upper cover of described main body cover gradually freezes save set with the mode of pressing and described blood bag is closely sealed.

6. blood bag according to claim 5 gradually freezes save set, it is characterized in that, the inner face periphery of described upper cover is provided with the annular ditch groove closely sealed with the opening periphery of described main body cover.

7. blood bag according to claim 1 gradually freezes save set, it is characterized in that, the main body that the upper cover of described main body cover gradually freezes save set with the mode fastened and described blood bag is closely sealed.

8. blood bag according to claim 7 gradually freezes save set, it is characterized in that, described main body cover and upper cover are respectively equipped with the exterior buckle type device of the mutual fastening of more than a group.

9. blood bag gradually freezes a using method for save set, it is characterized in that, comprises the following steps:

Cell culture or Cord blood are collected in freezer bag;

A wherein Teflon that agent has gradually been frozen in filling is gradually frozen bag to insert blood bag described in claim 1 and gradually freeze the bottom in save set main body;

Set up one deck blood bag in the top that described Teflon gradually freezes bag and insert space;

The freezer bag wherein comprising cell culture or Cord blood being inserted described blood bag inserts in space;

Place a wherein Teflon that agent has gradually been frozen in filling in the top that space inserted by described blood bag and gradually freeze bag;

Repeat described blood bag and described Teflon gradually to freeze the overlapping of bag and insert step;

The described blood bag completing assembling is gradually frozen save set sealing, within being placed in-80 DEG C 6-10 hour, make it lower the temperature gradually; And

The described blood bag of temperature being down to-80 DEG C gradually freezes save set and moves in liquid nitrogen and preserve.

10. method according to claim 9, is characterized in that, described blood bag is that temperature per minute declines 1 DEG C in the rate of temperature fall that described blood bag gradually freezes in save set; Described cell culture is stem cell culture.