TWI640630B - A freezing medium combination without fetal bovine serum (fbs) - Google Patents

Abstract

A cell cryopreservation composition without fetal bovine serum, which is mainly composed of a cryopreservation solution, A cryopreservation additive, human serum albumin, insulin, transferrin, 2-mercaptoethanol, and a saccharide, the cell cryopreservation composition without fetal bovine serum in cell recovery and inhibition of apoptosis/ The ability to necrosis is similar to and better than the conventional frozen cell preservation solution. In addition, the dosage of methyl guanidine (DMSO) commonly used in conventional frozen cell technology is reduced, and fetal bovine serum replacement is also caused. It is a human serum albumin, which reduces the side effects that thawing cells may cause when they are treated in humans.

Description

Cell cryopreservation composition without fetal bovine serum

The present invention relates to a frozen cell preservation composition, and more particularly to a cell cryopreservation composition which does not contain fetal bovine serum and which reduces the dose of methyl guanidine (DMSO).

The most common source of blood cells in the bone marrow is hematopoietic stem cells (HSCs). Hematopoietic stem cells have the ability to differentiate into various blood cells, which is very helpful for the recovery of blood cell functions of various lines. Blood-derived hematopoietic stem cells have a lower restriction of human leukocyte antigen matching and a greater number of hematopoietic stem cells; however, patients with chronic blood diseases are usually born for a long time after treatment. The cord blood is generally stored for a long time by cryopreservation technology, and the original activity of the hematopoietic stem cells can be preserved for future treatment.

The cell cryopreservation technique is to store the living cells in a liquid nitrogen (-195.79 ° C) after a stepwise cooling process. When necessary, the cells are activated by thawing procedures; It accounts for about 70% of the total volume, and it is easy to form ice crystals during the freezing process, and the ice crystal inside the cell has a strong killing power for the cells.

Too fast cooling rate will make the water inside the cell unable to pass out of the cell membrane through the cell membrane, which will cause the ice crystal inside the cell and the salt concentration to rise immediately, leading to cell death. Conversely, the temperature rising rate during thawing is too slow, which will make The water inside and outside the cell is repeatedly frozen and thawed. The ice crystals produced at this time will also cause damage to the cells. Therefore, when the cryopreservation technique is performed, an osmotic cryoprotectant is added to make the cells permeate and dehydrate, and the cryoprotectant replaces Water occupies the space inside the cell. At this time, the cell volume will shrink, and then the water will gradually reach equilibrium with the cryoprotectant into the cell, thereby protecting the frozen cells. The commonly used cryoprotectant includes: dimethyl Dimethyl sulfoxide (DMSO) and Fetal bovine serum (FBS).

Fetal bovine serum (FBS) is a serum made from the blood of unborn fetal calves. It has not been exposed to any pathogens in the environment and has not produced any antibodies in the blood. It has been widely used since the 1950s. The price is relatively expensive, but the fetal bovine serum contains various components, and the heterologous serum source may cause the risk of infection of potential animal pathogens, and the serum composition of each batch of fetal calves may be different, which may result in experimental results. The changes, in addition, the attachment factors in the serum may lead to immune responses of immune cells (such as macrophages and dendritic cells) and induce differentiation of stem cells... and so on.

Dimethyl sulfoxide (DMSO), a common amphiphilic molecule, is usually used as a solvent. It has very good antifreeze ability and is the most widely used cryoprotectant; but dimethyl hydrazine (DMSO) induces stem cell differentiation and causes mitochondria-associated apoptosis. If the thawed cells are applied to human health, it may cause other diseases in the patient. Therefore, the concentration of frozen cells should not be too high and frozen. Subsequent removal procedures must be true.

Human serum albumin (HSA), a human serum albumin synthesized by the liver, is a non-glycosylated plasma protein with various physiological functions. And a variety of biochemical properties, further buffering changes in pH and osmotic pressure, and assisting animal cells against mechanical shear stress.

Applicants have invented a frozen cell preservation composition which does not have fetal bovine serum (BSA) and which reduces the amount of dimethylammonium (DMSO) used, in view of the lack of prior art.

It is an object of the present invention to provide a cell cryopreservation composition which does not contain fetal bovine serum and which can reduce the dose of methyl guanidine (DMSO) to reduce the side effects which may occur when the frozen cells are thawed and subjected to human treatment.

For the above purposes, the technical means of the present invention consists in combining the following components into a cell cryopreservation composition consisting of: a cryopreservation solution, a cryopreservation additive, human serum albumin (HSA), insulin. , Transferrin, 2-Mercaptoethanol (2-ME), and a saccharide; wherein the cryopreservation composition may be dimethyl sulfoxide (DMSO), and the saccharide may be It is selected from any one of the following: Galactose, Lactose, Glucose, Sucrose, Mannose, Melibiose, and Trehalose.

The components of the cell cryopreservation composition can be produced by artificial synthesis to avoid the contamination of pathogens, and the effects of different substances in the batch production on subsequent experiments or treatments.

Fig. 1 is a result of cell proliferation after the first embodiment of the present invention.

Fig. 2 is a diagram showing the cell recovery rate at the time of thawing in the first embodiment of the present invention.

Figure 3 is a graph showing the total number of cells in the embodiment of the present invention.

Fig. 4 is a graph showing the results of apoptosis analysis of Example 1 of the present invention.

Fig. 5 is a second embodiment of the present invention, the proliferation ratio of CD34+/CD133+ before and after cell freezing.

Figure 6 is a graph showing the total number of CD34+/CD133+ cells in the second embodiment of the present invention.

The invention provides a cell cryopreservation composition, which comprises a cryopreservation solution with a concentration of 3% to 10%, a cryopreservation additive, a human serum albumin (HSA), and an insulin (Insulin). ), a transferrin (Transferrin), 2-mercaptoethanol (2-Mercaptoethanol, referred to as 2-ME), and a combination of sugars.

The human serum albumin (HSA) concentration is 1% to 5%, and the optimal concentration is 2%.

The concentration of the insulin (Insulin) was 1 μg/ml to 5 μg/ml, and the optimum concentration was 4.4 μg/ml.

The concentration of the transferrin (Transferrin) is 50 μg/ml to 100 μg/ml, and the optimal concentration is 60 μg/ml.

The concentration of the 2-mercaptoethanol (2-ME) was from 1 μg/ml to 5 μg/ml, and the optimum concentration was 2.02 μg/ml.

The concentration of the saccharide is 10 mM to 50 mM, and the optimal concentration is 30 mM, which may be selected from the group consisting of Galactose, Lactose, Glucose, Sucrose, and Mannose. (Mannose), melibiose, and trehalose, the most effective sugars are Galactose, Lactose, and Melibiose. The cryopreservation solution is 5% dimethyl hydrazine (DMSO), and the cryopreservation additive can be Iscove's Modified Dulbecco's Medium (IMDM). For the amount of use, please refer to the instructions provided by the purchase label.

The experiments will be divided into the following groups: control group, a conventional cold cell cryopreservation composition, consisting of: the cryopreservation additive, 10% dimethylarsine (DMSO), and 15% fetal bovine serum ( FBS).

The experimental group, that is, the cell cryopreservation composition of the present invention, is composed of: the cryopreservation additive, 5% dimethyl sulfoxide (DMSO), 2% human serum albumin (HAS), and 4.4 μg/ml insulin. (Insulin), 60 μg/ml Transferrin (Transferrin) and 2.02 μg/ml 2-mercaptoethanol (2-ME).

Control group: normal fresh cells that were not frozen, and normal culture was performed to compare cell growth after freezing/thawing.

Embodiment 1

Whether the cells were cryopreserved for the cells to protect the cells, and the cell recovery, the total number of cells, and the cell characteristics after thawing were analyzed.

Frozen cell

First, human hematopoietic stem cells (HSCs) proliferated for 7 days were centrifuged, the supernatant was removed, and the above-mentioned control group and the cell cryopreservation composition of the experimental group were placed in an ice bath to cool in an ice bath. In order to avoid damage to the cells after a large amount of heat is released after mixing with the cell liquid, the cell cryopreservation composition after the ice bath is uniformly mixed with the cells 1:1, and placed in a cryotube, the final cell density in the cryotube To maintain at 1 × 10 ⁶ cells / ml, then first place the cryotube into a constant-speed cooling box, and put the constant-speed cooling box into the -80 ° C refrigerator, let it cool down to the next day, and finally The cryotubes were transferred to a liquid nitrogen drum for cryopreservation.

Thawing cells

The cryotube was taken out from the liquid nitrogen, placed in a constant temperature water bath at 37 ° C for rapid thawing, and then a few cells were taken out to calculate the survival rate after cell freezing and the total number of viable cells, and then some cells were analyzed for apoptosis/ The proportion of necrosis.

Cell proliferation and cell viability

Please refer to Figures 1 to 3, the experimental group and the control group cell proliferation status is similar, and in the recovery rate and total cell test, the experimental group is better than the control group.

Apoptosis analysis (Annexin-V Assay)

This experiment uses the prior art PI and Annexin-V assay for analysis. The process is as follows: First, calculate the cell density using a conventional hemocytometer and WST-1 analysis, and take 1 × 10 ⁵ cells/vial to In a centrifuge tube, fill it up with PBS, centrifuge (2000 rpm, 10 min) and remove the supernatant liquid, then add 147 μl Annexin V binding buffer and 3 μl Annexin V-FITC fluorescent antibody, mix well and then protect from light (4 ° C , 10min), after the reaction, add 250μl Annexin Vbinding buffer, evenly mix and centrifuge (2000rpm, 10min), remove the excess fluorescent antibody, after centrifugation, remove the supernatant liquid, add 144μl Annexin V binding buffer and 6μl PI The fluorescent dye was uniformly mixed and then protected from light (4 ° C, 10 min). Finally, an Accuri C6 flow cytometer was used for analysis.

When the cells are in early apoptosis, the phospholipidin (PS) in the cell membrane is turned outward from the inner side of the cell membrane to the outer side of the cell membrane, while the Annexin V protein has a high affinity for the phospholipid serine, so it is utilized. The annexin V is connected to the fluorescent substance to detect whether the cells have apoptosis.

Please refer to Figure 4, PI and Annexin-V analysis will show four quadrants. In the upper left of the experiment, PI has a signal, and Annexin V has no signal. This quadrant is due to external environmental factors leading to cell death (Necrosis ), the upper right is PI and Annexin V have signals, which means the end of Apoptosis, the bottom left is PI and Annexin V have no signal, which means normal cells, while the lower right is PI without signal, but Annexin V has signal, which means Cell apoptosis for Apoptosis

Please refer to Fig. 4 again, the Apoctotic rate of the experimental group was 17.3% to 23.3%, and the control group was 23.7%. From the results of the Annexin V/PI analysis, the frozen cell preservation combination of the present invention is known. The cells still have good recovery and proliferation ability after freezing and thawing the cells, and therefore, the cell cryopreservation composition of the present invention can replace the conventional cryopreservation composition.

Embodiment 2

It is identified whether the frozen cells still have the basic characteristics of hematopoietic stem cells.

Cell community formation experiment

The cells were analyzed by Colony-Forming Units (CFU Assay), and the thawed cells were seeded in semi-solid medium (MethoCultTM GF H4434) and cultured in a wet CO ₂ incubator for 10 to 14 days, followed by An inverted microscope observes and records that if cells can form a community of different blood cell precursor cells, it means that these cells have the ability to further differentiate into specific blood cells.

Table 1 shows the number of colonies that can be formed per 200 cells in total cells. The results show that although the experimental group formation ratio is slightly lower, the results show that the frozen hematopoietic stem cells still have the ability to form colonies.

Cell Surface Antigen Analysis

Clinically, in order to quickly determine whether the amount and characteristics of cells are suitable for transplantation, it is generally judged whether or not the indicator of hematopoietic stem cells is based on the expression of cell surface antigens, and the most commonly determined antigen is CD34. After long-term research, it was found that the surface antigen expression of hematopoietic stem cells should have both CD34, CD90, CD133, and cells without CD38 surface antigen.

In this experiment, standard double staining was performed on CD34 and CD133 antigens. After calculating the cell density, 8 x 10 ⁴ cells/vial was taken to 5 ml polystyrene test tube, and PBS was added to full, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. Then, add 2 μl of each of CD34-FITC and CD133-PE fluorescent antibody, mix well and avoid the light reaction at 4 ° C for 30 min; add PBS to full volume, centrifuge, and discard the supernatant; finally, flow with Accuri C6 Cytometry analysis.

Referring to Fig. 5, the CD34+/CD133+ cells in the control group increased by about 2.7 times after three days of proliferation, while the experimental group increased by about 2.15 times, and the experimental group showed a near-controllity compared with the control group.

Please refer to Fig. 6 to freeze the total cells of 1 x 10 ⁶ cells at the same time, and in the condition of all kinds of thawing after thawing, a total of 2.5 x 10 ⁵ CD34+/CD133+ cells in the control group after three days of proliferation, The group was 1.84 x 10 ⁵ CD34+/CD133+ cells, which was much higher than the control group.

In summary, the components of the frozen cell preservation composition of the present invention can be produced by artificial synthesis to avoid the contamination of pathogens, and the effects of different substances in the batch production on subsequent experiments or treatments. After the cell is frozen and thawed, the composition of the present invention still has good cell recovery and community ability for hematopoietic stem cells, and preserves a special protein on the surface of the hematopoietic stem cell; in addition, the dimethyl group is lowered in the composition of the present invention. The use of guanidine (DMSO) and the replacement of fetal bovine serum (FBS) with human serum albumin (HSA) will reduce the side effects of thawing cells during human treatment and reduce cell freezing. The damage suffered.

Claims (2)

A cell cryopreservation composition for cell therapy without fetal bovine serum is a concentration of 5% dimethyl hydrazine (DMSO), a cryopreservation additive Iscove's Modified Dulbecco's Medium (IMDM), a concentration 2% human serum albumin (Human serum albumin), a concentration of 1 μg / ml ~ 5 μg / ml of insulin (Insulin), a concentration of 50 μg / ml ~ 100 μg / ml of transferrin (Transferrin), a concentration It is composed of 1 μg/ml to 5 μg/ml of 2-mercaptoethanol and a concentration of 30 mM sugar. The cell cryopreservation composition for fetal cell serum for cell therapy according to claim 1, wherein the saccharide is selected from the group consisting of galactose, lactose, and glucose (Glucose). Any of sucrose, Mannose, melibiose, and trehalose, and most preferably galactose, lactose, and melibiose.