KR100522458B1 - The Thawing Connective Instrument and Thawing Method for Cryopreserved Umbilical Cord Blood Cells using the Thawing Connective Instrument - Google Patents

Abstract

The present invention removes DMSO, a toxic component added to increase umbilical cord blood cell cryopreservation during cryopreservation, and maximizes the recovery of viable cells during thawing to thaw cryopreserved umbilical cord blood cells to increase the likelihood of successful umbilical cord blood cell transplantation. It relates to a connection mechanism and a thawing method using the same.

Specifically, thawing cord blood cells frozen in the freezing bag using a thawing solution consisting of 5% Dextran-40 + 5% albumin formed by mixing 10% Dextran-40 and 20% human serum albumin, It is characterized by providing a connection mechanism for thawing that is installed to supply.

In addition, it is characterized by providing a method for thawing cord blood cells using the thawing solution and the thawing coupling mechanism.

Accordingly, by thawing under the closed-circuit system as described above, cryopreserved umbilical cord blood cells can be thawed aseptically to recover more nucleated cells more stably, and show higher cell viability. To make it visible.

Description

The Thawing Connective Instrument and Thawing Method for Cryopreserved Umbilical Cord Blood Cells using the Thawing Connective Instrument}

The present invention relates to a thawing connection apparatus and a thawing method using the same, which are used for transplanting umbilical cord blood cells that are freeze-stored in liquid nitrogen after separating nucleated cells from the collected placenta and umbilical cord blood. A thawing connection device for connecting between the freezing bag and the transplantation bag for umbilical cord blood storage and the thawing connection device were used to introduce lysed cord blood cell thaw solution consisting of Dextran-40 and human serum albumin to induce umbilical cord blood. It is about thawing method to thaw.

In general, cryopreservation of tissues and cells at cryogenic temperatures has been in use since the 1700s, and rapidly developed since the mid-1900s when the effects of anti-freezing agents added to cell cryopreservatives were discovered.

At present, animals and human cells, tissues, sperm, eggs, embryos and the like are routinely frozen.

At this time, the basic principle of the cryobiology is applied irrespective of the type of cells to be frozen.

Before freezing, cells were treated with cryopreservatives such as glycerol, dimethyl sulfoxide (DMSO), or ethylene glycol to enhance the cryopreservation of the cells.

At this time, DMSO (Dimethyl sulfoxide), the main component of the cryopreservative, has a significant effect on the survival rate of cells required for hematopoietic stem cell transplantation as the frozen cells undergo thawing.

In addition, the DMSO is a cryopreservative used to maintain cell viability even at very low temperatures, and has a disadvantage of toxicity to cells when the temperature is maintained at 37 ° C.

In addition, the DMSO has a characteristic of inducing a sudden fluid shift and cell death when the maintenance temperature reaches 37 ° C. while creating a hypertonic environment in the cell, and the destruction of red blood cells causes accumulation of free hemoglobin outside the cell. In addition, if it is introduced into the vein will cause neurotoxic effects.

In addition, the DMSO causes adverse effects such as blood pressure instability, fever, chills, and nausea when introduced into a living body.

In order to solve these problems, conventionally, a solution was solved by thawing the cryopreserved cells and immediately mixing them with a high-performance solution of Dextran 40 + 5% albumin, followed by washing the thawed cells and DMSO, The procedure to remove free hemoglobin, cell product, etc., which is a product of erythrocyte destruction, and several procedures before administering hematopoietic stem cells to the patient were performed.

In addition, the nucleated cells isolated from the collected placenta and cord blood are mostly frozen with 10% DMSO before storage using a cryopreservative added with DMSO, and the thawing solution and thawing used above the thawed nucleated cells. There are many ways.

According to one of such thawing methods (Rubenstein et al. , Proc Nat Acad Sci USA, 92: 10119-10122, 1995), the thawing solution is a saline solution containing 5% Dextran 40 and 2.5% Human albumin. In the thawing process, a method of recovering the cells by centrifugation at 400 g for sedimentation is known. As a result, nucleated cells were high as 94.46%, but the viability of viable cells was reduced to 63.3%.

An object of the present invention for improving the above problems, to easily remove the toxic components DMSO added to increase the cryopreservation of umbilical cord blood cells during cryopreservation process, and maximize the recovery of viable cells during thawing Increase the chance of blood cell transplant success, remove DMSO contained in cryopreservative, maximize recovery of viable cells, make operation simple, and work in closed circuit to prevent invasion of bacteria To provide a cryopreserved umbilical cord blood cell thawing connection mechanism and a thawing method using the same.

In order to achieve the above object, the present invention is configured as a thawing unit connected to form a closed circuit in a cord blood cell freezing bag and a bag for transplantation, wherein the thawing unit is connected to a main pipe line and both ends thereof, and the distribution port. It is characterized in that it provides a thawing connection mechanism which is installed to supply to the transplant bag after thawing the cord blood cells frozen in the freeze bag made of a transport pipe connected to each.

In addition, the present invention prepares a thawing solution consisting of 5% Dextran-40 + 5% albumin formed by mixing 10% Dextran-40 and 20% human serum albumin to a thawing unit in which a freeze bag and a transplant bag are connected in a closed circuit. Injecting; Washing DMSO simultaneously with thawing of the freeze bag; Centrifuging to make sediment of cord blood cells; Characterized by providing a method of thawing umbilical cord blood cells using a thawing coupling device comprising the step of preparing the cells for transplantation by removing the supernatant and resuspension.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 to 4, the thawing connection mechanism of the present invention is configured as a thawing unit 200 which is connected to form a closed circuit between the umbilical cord blood cell innocence 100 and the transplantation bag 300.

The thawing unit 200 is configured as a main pipe 210 and distribution ports 212 and 214 connected to both ends thereof, and the distribution port 214 includes a main bag 130 and a sub bag 110. A plurality of transfer pipes 232 and 234 for connecting with the umbilical cord cell innocence 100 is made, respectively, and spikes 232b and 234b are connected to the end thereof.

 Dispensing port 212 is connected to the lower end of the main pipe line 210 is also connected to the transfer pipe line 252 (254) is connected to the needle 252b and the cylinder port 254b is connected to the end.

In addition, clamps 232a, 234a, 252a, and 254a are fixed to one side of the transfer pipes 232, 234, 252, and 254 connected to the distribution ports 212 and 214, respectively.

Meanwhile, as shown in FIGS. 3 and 4, the thawing unit 200 of the present invention includes a check valve 216 in the delivery pipe 254 into which the thawing liquid 400 is injected from the distribution port 212. Pressure valves 218 are respectively installed in the transfer pipe 252 connected to the implanted bag 300.

The pressure valve and the check valve may include balls 216b and 218b for blocking a pipe and springs 216a and 218a installed to give elasticity to one side thereof.

Referring to the operation of the thawing mechanism of the present invention having the above configuration as follows.

As shown in Figures 1 to 4, by connecting the thawing unit 200 between the umbilical cord blood cell innocent 100 and the transplantation bag 300 to form a closed circuit, the frozen cells are in contact with the outside air It is thawed without being injected into the graft bag 300.

At this time, the thawing unit 200 is a feed line 232 and 234 and the needle 252b and the cylinder port 254b to which the spikes 232b and 234b are connected, respectively. Distributing openings 212 and 214 of both ends are formed through a main pipe line 210 is connected to each other to prevent contact with the outside air.

In addition, clamps are respectively fixed to the transfer lines 232, 234, 252, and 254 to manually control the flow of the fluid.

The thawing of the cells through the closed conduit of the same thawing unit having the same configuration as described above is clamped to the delivery conduits 254, 232 and 234 while injecting the thawing liquid through the cylinder port 254b. Removing 254a), the thawing fluid is injected into the cord blood cell innocence 100 through the spike.

At this time, the umbilical cord blood cell copper white (100), the normal bag consisting of the main bag 130 and the sub-bag (110) is thawed at a predetermined temperature while thawing fluid is injected.

Then, when the thawing of the cord blood cell innocence 100 is completed, the clamp 254a is fixed to the transfer pipe 254 and then the clamp 252a of the other transfer pipe 252 is removed, and thawed by the operation. Cells are injected into the graft bag 300.

On the other hand, as shown in Figures 3 and 4, the present invention is automatically opened by the operation of the pressure valve 218 and the check valve 216 without the need for fixing and separating the clamps respectively fixed to the conveying line. And thawing the cells of the cord blood cell copper whitening (100) while closing to inject into the graft bag (300).

As described above, the operation of the thawing unit 200 having the pressure valve 218 and the check valve 216 is first performed by injecting the thawing liquid through the cylinder port 254b of the feed pipe 254. The valve 216 opens and is injected in only one direction.

Then, the injected thawing solution is injected into the cord blood cell copper infusion 100 through the spikes 232b and 234b connected to the ends of the transfer lines 232 and 234.

Subsequently, when the thawing of the cord blood cell innocence 100 is completed, when the pressure is applied to the freezing bag 100, the pressure valve 218 installed in the branch pipe 212 is the inside of the freezing bag 100. The thawed cells are automatically opened by the pressure and injected into the graft bag 300.

At this time, the inflow to the feed pipe 254 side by the check valve 216 is prevented, it is easy to operate at the pressure required by the tension of the spring of the pressure valve 218.

Meanwhile, the thawing solution of the present invention consists of 5% Dextran-40 + 5% albumin formed by mixing 10% Dextran-40 and 20% human serum albumin.

At this time, the Dextran40 and albumin are used to maintain a proper osmotic pressure in vivo as a general fluid, the reason for using 5% Dextran and 5% albumin because it has a colloidal osmotic pressure, such as plasma, frozen during thawing By maintaining the osmotic pressure with the cells to minimize the damage to the cells.

In addition, the thawing method of the present invention through a thawing unit that is connected to the thawing solution and the umbilical cord blood cell innocence and a transplantation bag to be closed in the closed circuit to thaw umbilical cord blood cells thawed cytotoxic component DMSO .

First, as shown in FIG. 5, Dextran 40 (USP) is dissolved in physiological saline to prepare 10% Dextran 40.

Then, 10% Dextran-40 and 20% human serum albumin are mixed to prepare 5% Dextran-40 + 5% albumin.

Next, a thawing unit for preparing the thawing solution into the cord blood cell whitening in a closed circuit is prepared, and the prepared thawing solution is divided into a thawing solution for washing and a resuspension thawing solution.

Subsequently, it forms a conduit connected to the graft bag and the cord blood cell innocence, and the thawing solution injection conduit is connected to one side thereof so that the thawed solution is injected into the cord blood cell innocence and then directed to the graft bag. The thawing liquid is injected through the thawing unit 200 installed.

The thawing unit is prepared by injecting the thawing liquid into the cylinder port 254b, and preparing the input of the thawing unit to connect the spike and the needle of the thawing unit to the cord blood cell innocence and the transplanting bag to prevent the introduction of bacteria and the like.

At this time, a clamp is prepared in advance in the transfer pipe to which the spike and the needle are connected to facilitate the closing of the pipe.

Then, the transfer pipe connected to the graft bag is locked with a clamp, and the thawing solution is prepared to flow only inside the cord blood cell innocence.

Subsequently, the frozen bag containing the nucleated cells in the cord blood is dissolved in a 37 ° C. temperature controlled bath until the cord blood nucleated cell freeze reaches a mixture of ice and liquid form.

Then, in order to remove the DMSO, a cryopreservative component of the cryopreserved cord blood, the thawing liquid is introduced into the freezing bag through the cylinder port of the thawing unit, and mixed to completely mix the lysate and the thawing solution in the freezing bag.

The freezer and the freeze bag are mixed for at least 4-5 minutes so that the cord blood cells are completely mixed with the thawing solution.

Subsequently, when the thawing solution and cord blood cells are mixed in the freezing solution, the transplant bag wrapped in the ice bag is down, and the freezing bag surrounded by the ice bag is up, and then the clamp is fixed to the delivery line. Introduce this prepared graft bag.

Then, centrifugation is performed to make a precipitate of cells introduced into the graft bag, which is centrifuged at 1000 g for 15 minutes in a 4 ° C centrifuge to settle the cells.

The cells are then resuspended as well as the supernatant removed from the settling for preparation of the cells for transplantation.

In the above operation, after centrifugation, the transplantation bag is carefully connected to a plasma extractor, the thawing solution in the implantation bag is removed with a waste bag, and the cell layer at the bottom of the implantation bag is suspended with a resuspension thaw solution. .

As a result of thawing the cryopreserved umbilical cord blood frozen and stored in liquid nitrogen under a closed circuit system using a thawing solution and a thawing apparatus set, the final total nucleated cell recovery was 92.70 ± 1.32% and the cell viability was 86.38. Nucleated cell recovery and cell viability were high as ± 3.17%.

In addition, umbilical cord blood cells were collected from the frozen storage, and samples were taken immediately after thawing in a 37 ° C. temperature-controlled bath. Nucleated cells were counted using an automatic hemocytometer and 0.4% Trypan blue cells under an optical microscope. As a result of measuring the survival rate, 97.97 ± 1.32% of the nucleated cells compared to the number of nucleated cells measured before cryopreservation were recovered and the survival rate of the cells was 88.41 ± 0.75%.

Then, the thawing solution for washing was added to the freezing bag, and the cell suspension was transferred to the graft bag containing the thawing solution, and the nucleated cell recovery and cell viability in the graft bag were measured, and 94.59 ± 3.73% and 88.41 ± 3.16, respectively. Showed%.

Finally, the number of nucleated cells in the waste fluid was measured by immersing the cells through centrifugation and removing the supernatant with the cryopreservative added (Table 1).

TABLE 1

Thawing stage Right after thawing After adding thawing solution Resuspend Cell survival rate 88.41 ± 0.75 88.04 ± 3.16 86.38 ± 3.17 Nucleated Cell Recovery 97.97 ± 1.32 94.59 ± 3.73 92.70 ± 2.72

After thawing the cryopreserved cord blood stored frozen by the closed circuit thawing unit according to the present invention, the aseptic state of the thawed cells was confirmed.

As a result, as shown in Table 2, the umbilical cord blood, thawed solution, and samples for each step were sterile-tested (aerobic bacteria, aerobic fungi, anaerobic bacteria, anaerobic fungi) before the cryopreservation.

TABLE 2

Inspection Cord blood Thawing solution Waste Resuspend result Not detected Not detected Not detected Not detected

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

[Example 1] Thawed cord blood thawed using a thawing solution and a set of thawing connection apparatus

<1-1> Recovery of cryopreserved cord blood stored frozen in liquid nitrogen

The collected nucleated cells in the placenta and umbilical cord blood were added to cryopreservative, 50% DMSO + 5% Dextran-40, and stored in BioArch ^™ , a cryopreserved product manufactured by ThermoGenesis, and recovered according to the recovery procedure.

<1-2> 10% Dextran-40 manufacture

In a sterile workbench, sterile 500ml beaker is poured into 150ml of saline solution, 30g of Dextran 40 (USP) is placed on top of saline solution and allowed to stand at room temperature for 16 hours to dissolve. 10% Dextran 40 was prepared.

<1-3> thawing solution

225 ml of 10% Dextran-40 and 75 ml of 20% albumin were mixed and filtered aseptically by 0.45 μm and 0.22 μm filters.

Finally, the prepared thawing solution was 5% Dextran-40 + 5% albumin.

<1-4> thawing solution set preparation

Prepare the thawing solution 75ml of the thawing solution prepared by putting 25ml of thawing solution in a graft bag and two 30cc syringes, and preparing a resuspension thawing solution containing the thawing solution in a 50cc syringe after the completion of thawing. It was.

<1-5> Closed Circuit System Preparation

In a sterile workbench, aseptically connect the syringe port of the graft bag to the needle of the thawing connector set, lock the clamps of the graft bag and the instrument set, and place the bag in a 4 ° C ice bag to keep the thawing solution cool. Put in.

Washing and thawing solutions in syringes were also refrigerated until use to keep them cool.

<1-6> Thawed Blood Frozen

The canister of cryopreserved cord blood recovered from BioArchive ^™ was opened, and the overlap bag that packed the frozen bag was disinfected with 10% bridge solution and placed in a zipper bag.

Umbilical cord blood nucleated cell lysates contained in the zipper bag were dissolved in a 37 ° C. temperature controlled bath until the ice and liquid form reached a mixed state.

The zipper bag containing the cord blood nucleated cells was removed from the temperature control tank, disinfected on the outside and placed on a sterile workbench.

The freezing bag was removed from the zipper bag of the sterile workbench, the overlap bag was removed, and if necessary the outer surface of the freezing bag was dried.

<1-7> Closed Circuit System Establishment

The pot surface of the freezing bag was wiped clean with 10% bridging solution and the top cap was cut.

The cut and inner surface were cleaned with alcohol.

Two spikes connected to the spike line of the thawing connector set were inserted into the freezer bag until the diaphragm was punctured and placed in an ice bucket.

At this time, the spike was inserted deep until the diaphragm of the freeze bag was opened. Otherwise, the thawing solution may leak out when the thawing solution is injected.

<1-8> DMSO Cleaning Process

A syringe containing 25 ml of thawing solution was inserted into the syringe port of the thawing connector set, and the thawing solution was introduced into the freezing bag for 1-2 minutes, and the mixture was mixed well by hand to completely mix the frozen cell and the thawing solution in the freezing bag. .

At this time, only the clamp of the part connected with the implant bag should be locked and all other clamps should be open.

The ice pack was sized so that the freezing bag could expand by the amount added.

Mix for at least 4-5 minutes to completely mix the thawing solution with the cells.

After thoroughly mixing, the transplant bag wrapped in the ice bag was down, and the freezing bag surrounded by the ice bag was raised up to allow the cell suspension to flow into the transplant bag.

At this time, only the clamp of the syringe port in the thawing connector set was locked and the remaining clamps were opened.

The clamp of the implant bag was locked to clean the freezer bag.

A syringe containing 25 ml of thawing solution was inserted into the syringe port of the thawing apparatus set to introduce thawing solution into the freeze bag.

The freezing bag was shaken to mix the remaining cell suspension with the thawing solution to mix the solution in the bag.

The clamp of the syringe port was locked and the clamp opened between the freezing bag and the implantation bag and the wash solution and cell suspension were transferred from the freezing bag to the implantation bag.

This process was repeated twice.

The total amount of the graft bag was about 150 ml (75 ml of transplantable bag thawing solution + 25 ml of cell suspension in cryopreservative + 1, 25 ml + 2, 25 ml).

The total amount in the graft bag was not more than 175 ml to prevent breakage during centrifugation.

<1-9> Centrifugation to make cell precipitate

The transplantation bag, thawing connector set and freeze bag were separated and the implantation bag was placed in a centrifuge bucket.

The bag stood up with the port facing upwards.

Weigh and balance. The second bucket is balanced.

Cells were immersed by centrifugation at 1000 g for 15 minutes in a 4 ° C centrifuge.

<1-10> Supernatant Removal and Resuspension for Cell Preparation for Transplantation

The cell transplant bag was removed after centrifugation, taking care not to resuspend the cell layer at the bottom of the implant bag.

The implantable bag was installed in the plasma extractor, taking care not to suspend the cell layer at the bottom of the bag.

The syringe of the implantable bag was inserted into the port of the disposal bag.

The 130 ml thawing solution was transferred from the implantation bag to the waste bag and then removed.

Pressure was applied until the cell layer at the bottom of the implant bag was suspended or until the remaining cell suspension reached about 20 ml.

The cells in the remaining cell suspension were well suspended. The desired amount of thawing solution in the implant bag was introduced using two syringes containing 40 ml of thawing solution and suspended well (desired amount between 30 and 150 ml).

As described above, after freezing and storing the nucleated cells separated in the placenta and cord blood collected according to the method of the present invention, the thawing method under a closed circuit system using a thawing solution, a thawing connector set, a transplantation bag When thawing and implanting cryopreserved umbilical cord blood cells, they can maintain a sterile state, recover more nucleated cells, and show higher cell viability.

While the invention has been shown and described with respect to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention as provided by the following claims. I would like to clarify that those who have knowledge of this can easily know.

1 is a perspective view illustrating a state in which a freezing bag, a transplantation bag, and a waste bag are connected by using a thawing connection mechanism according to the present invention.

Figure 2 is a perspective view showing a connection mechanism for thawing umbilical cord blood cells according to the present invention.

3 is a perspective view showing a thawing apparatus for umbilical cord blood cell thawing according to another embodiment of the present invention.

4A and 4B are cross-sectional views illustrating a flow state of injection of thawing solution in FIG. 3, respectively.

Figure 5 is a flow chart illustrating a cryopreserved cord blood cell thawing method frozen in accordance with the present invention.

Figure 6 is a graph illustrating the analysis of viability of nucleated cells in each stage of thawing cryopreserved umbilical cord blood cells in the present invention.

Figure 7 is a graph analyzing the change in cell recovery of each step of thawing cryopreserved cord blood cells in the present invention.

* Description of the symbols for the main parts of the drawings *

100 Umbilical cord blood cell innocence 210 Main passage

214 Dispensing port 218 Pressure valve

252 .. 300 Songsong-ro

Claims (10)

The thawing unit 200 is connected to form a closed conduit between the cord blood cell innocence (100) and the transplantation bag (300), The thawing unit 200, It is configured as the main conduit 210 and the distribution ports 212, 214 connected to both ends thereof, The distribution port 214 is connected to a plurality of transport lines 232 and 234 for connecting with the cord blood cell copper innocence 100, respectively, The transfer pipes 232, 234, 252, 254 is provided with a clamp on one side to manually open and close it, A pressure valve 218 and a check valve 216 formed of balls 216a and 218a which block the conduit and springs 216a and 218a installed to give elasticity to one side thereof are the transfer pipes 252 and 254. Installed on each) Dispensing port 212 is connected to the lower end of the main pipe line 210, the transfer pipe line 252, 254 is connected to the graft bag 200 and the cylinder port 254b for injection of the thawing solution, Spikes 232b and 234b are connected to the end of the transport line 232 and 234 connected to the cord blood cell innocence 100 and the end of the transport line 252 connected to the transplant bag 200. Freezing storage cord blood cells thawing connection mechanism consisting of a configuration in which the needle (252b) is connected. delete delete delete delete Mixing 10% Dextran-40 and 20% human serum albumin to form 5% Dextran-40 + 5% albumin, which is a thawing solution; After forming a conduit connected to each of the transplant bag and the cord blood copper ingot, connect the thawing agent infusion pipe so that only one side of the thawing fluid can be injected from one side, and the thawing solution is injected into the cord blood copper inoculation. Installing a thawing connection mechanism to direct only to the transplant bag after injection to form a hermetic conduit between the umbilical cord cell innocence and the transplant bag; Thawing the stored cord blood in a bath maintained at 37 ° C. so that the cord blood nucleated cell lysate reaches a mixed state of ice and liquid; Injecting thawing solution into the frozen cells through a hermetically sealed tube and mixing the thawing solution; Centrifuging the thawed cells for 15 minutes in a centrifuge at 4 ℃ to form a precipitate; And Thawing method of cryopreserved umbilical cord blood cells, comprising the step of removing and resuspending the supernatant for the preparation of cells for transplantation. delete delete delete delete