TWI643953B - Method for culturing progenitor cells by autologous plasma - Google Patents

Abstract

The present invention provides a method for culturing protocells in autologous plasma, comprising: preparing a raw cell; taking a culture solution; and adding an additive comprising 1% to 10% by volume of autologous plasma to the original cell And cultivated. Wherein the autologous plasma is obtained by centrifuging autologous blood twice; and filtering with a filter to obtain autologous plasma for cell culture. Wherein the supplement further comprises from 0.5 ng/mL to 10 ng/mL recombinant human basic fibroblast growth factor and from 1 U/mL to 10 U/mL heparin. The invention can successfully use the limited autologous plasma to culture the original cells, and the desired number of cells can be obtained, and the autologous plasma can be prepared in time and the preparation time is short, so as to facilitate the cultivation of the original cells.

Description

Method for culturing primitive cells with autologous plasma

The present invention relates to a method of culturing primordial cells, and more particularly to a method of culturing primordial cells in autologous plasma.

Serum is the most widely used additive in cell culture because it provides all the nutrients needed for cell growth. Serum from animals, especially fetal bovine serum, will be rigorously tested in clinical applications because of the possibility of importing foreign sources and animal proteins may cause adverse reactions to cells. Therefore, there is a growing trend to switch to chemically defined supplements (chemically defined supplements). Serum is still the best choice because chemically defined media does not always achieve the desired results.

In the prior art, the plasma for cell culture is prepared by first separating the plasma, dialysis for 12 hours to 16 hours overnight, dialysis for 4 hours, and finally centrifuging for 15 minutes, and then filtering the supernatant for about 16 hours to 20 hours. . This method can be applied to allogeneic and mixed plasmas of different donors, but the method of preparing autologous plasma and applying it to cell culture is not as effective as expected. First, the autologous plasma obtained by applying this dialysis method has a small number of cells after being cultured by adding 20% of autologous plasma. The number of cells in the P0 generation (culture day 17) was 1.56× ^{10 6} , the number of cells in the P1 generation (culture day 24) was 2.1× ^{10 7} , and the number of cells in the P2 generation (culture 31 days) was 6.04× ^{10 8} and P3. Generation (Day 39 cultures) cell number 1.1x10 ¹⁰ th. Secondly, the plasma preparation method takes too long and takes about 16 hours to 20 hours, which cannot be obtained in time when needed.

In addition, the use of autologous plasma/serum to cellular products will be the least risky option for the risk of importing foreign pathogens and foreign substances once the cell product is to be used in the clinic. In order to reduce the risk of the above-mentioned foreign pathogenic bacteria and foreign substances in the culture of human cells, if the autologous cells can be cultured in autologous serum/plasma, the utilization, application and safety of autologous cells can be greatly improved. Therefore, it is urgent to propose an autologous serum/plasma for autologous cell culture in order to safely use cell therapy and develop regenerative medicine.

In order to overcome the disadvantages of the prior art, the method of the present invention is to use autologous plasma to culture autologous progenitor cells, preferably autologous umbilical cord primitive cells, which are derived from The cord blood stores the remaining autologous cord blood plasma (CBP) after storage, and can be obtained in time for the umbilical cord original cell culture.

In order to achieve the above object, the technical means adopted by the present invention is to provide a method for culturing original cells in autologous plasma, comprising: preparing a raw cell; taking a culture solution; and containing a volume percentage of 1% to 10% An addition of % autologous plasma is added to the original cells and cultured.

"Autologous plasma" as used in the present invention means the plasma and desire The cultured primordial cells are all derived from the same individual, and autologous plasma is used for the culture of autologous primitive cells.

Preferably, the original cell is an umbilical cord primitive cell.

Preferably, the culture solution comprises minimum essential medium (MEM), α-MEM medium, basal media eagle (BME), DMEM medium (Dulbecco's modified Eagle's medium, DMEM), MEM/ F12 medium, Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium (RPMI), Medium 199 medium, or a combination thereof.

Preferably, the autologous plasma system comprises the steps of: centrifuging autologous blood twice; and filtering with a filter to obtain autologous plasma for primordial cell culture.

Preferably, in the step of centrifuging autologous blood twice, the first centrifugation speed is from 400 g to 500 g, and centrifugation is performed for 5 minutes.

Preferably, in the step of centrifuging autologous blood twice, the second centrifugation speed is from 1000 g to 1500 g, and centrifugation is performed for 15 minutes.

Preferably, in the step of filtering by the filter, the filter is a 0.22 micrometer (μm) filter.

Preferably, the volume percentage of the autologous plasma is 5%.

Preferably, the additive further comprises a growth factor.

More preferably, the growth factor is recombinant human fibroblast growth factor-basic (rh-bFGF) at a concentration of 0.5 ng/mL per ml. Up to 10 ng/mL.

More preferably, the recombinant human basic fibroblast is produced The concentration of the long factor is 1 ng/mL.

Preferably, the additive further comprises an anticoagulant.

More preferably, the anticoagulant is heparin and the concentration is from 1 U/mL to 10 U/mL.

More preferably, the concentration of the heparin is 2 U/mL.

Preferably, the autologous plasma is autologous umbilical cord plasma.

In general, various media components are enriched in four basic elements, including amino acids, carbohydrates, inorganic salts and vitamins. Therefore, the culture solution used in the method of culturing the original cells by using the autologous plasma prepared by the above method may further comprise a nutrient additive which promotes growth of the original cells, and may be applied to the nutrition in the present invention. Additives include, but are not limited to, hormones, proteins, biohormones, antibiotics, and growth factors. The above-mentioned growth factor which can be applied to the present invention can be used in the present invention as long as it is any growth factor which can be applied to cell culture and can be used to promote growth of primordial cells, and the growth factor contains, but does not Limited to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), recombinant human fibroblast growth factor-basic (rh-bFGF) and Recombinant human insulin growth factor (Insulin-like growth factor 1, IGF-1).

The invention has the advantages that the original cells can be successfully cultured by using limited autologous plasma, and the required number of cells can be obtained, and the autologous plasma can be prepared in time and has a short preparation time to facilitate the cultivation of the original cells.

Figure 1 shows the number of cells after different cultures were added by adding different additives to the α-MEM medium. The first group of culture medium contains 10% fetal bovine serum (FBS); the second group contains 5% autologous cord blood plasma (CBP), 1ng/mL recombinant human alkaline Fibroblast growth factor and 2U/mL heparin; the third group contains 5% highly concentrated platelet lysate and 2U/mL heparin; the fourth group contains 10% concentrated platelet lysate and 2U /mL heparin.

The invention is further illustrated by the following examples which are not intended to limit the invention. A person skilled in the art can make some modifications and modifications without departing from the scope of the invention.

Preparation 1 Separation of umbilical cord progenitor cells

The umbilical cord was first immersed in 75% alcohol for about 20 to 30 seconds and then transferred to a petri dish containing 1x phosphate buffered saline (PBS). Next, the umbilical cord is cut for about 3 to 4 sections, and each section is moved to a clean sterile Petri dish. Place the tip of the scissors into the ostium and cut it along the vein, and pull the umbilical cord apart with a forceps.

The two arteries were carefully peeled off with forceps and any blood was prevented from splashing onto the Wharton's jelly of the umbilical cord connective tissue, followed by removal of the vein. Carefully remove the Walden gum from the remaining umbilical cord tissue and move it Keep in a petri dish containing a little a-MEM medium to keep it moist. Then, the F. chinensis gel was cut into finely divided pieces by a surgical scissors, and the chopped Wyner's gum was evenly divided into 4 to 6 petri dishes and 10 ml (mL) of α-MEM culture solution was added. These petri dishes containing Walden gum were placed in a 5% carbon dioxide incubator at 37 ° C for 1 night, preferably 12 to 16 hours, to obtain umbilical cord original cells.

Preparation 2 Preparation of autologous umbilical cord plasma for addition

Blood was taken from the two arteries peeled off during the operation of Preparation Example 1 and the removed vein, and secondary centrifugation was performed. The first centrifugal speed is 400g to 500g (low speed centrifugation), centrifugation for 5 minutes; the supernatant is taken and then subjected to a second centrifugation, the rotation speed is 1000g to 1500g (high speed centrifugation), the supernatant is taken for 15 minutes, and The resulting plasma has a platelet content ranging from ⁴ x ^{10 4} /μl to 14 x 10 ⁴ /μl. The autologous umbilical cord plasma was then filtered through a 0.22 μm filter for sterility, and then the filtered autologous umbilical cord plasma and 2 U/mL heparin were added to the culture dish containing the autologous umbilical cord original cells of Preparation Example 1.

Preparation Example 3 Cell Culture

Will contain 10 ml of growth medium (containing α-MEM, 5% autologous umbilical cord plasma, 1 ng/mL recombinant human basic fibroblast growth factor and 2 U/mL heparin) and the autologous umbilical cord blast cells of Preparation Example 1 The culture dish is placed in a carbon dioxide incubator for cultivation. 3 ml of growth medium was added every 3 days. After 10 days, the Walden gum pieces were removed, and the cells were washed with 1 time PBS and then the growth medium was added. Subculture was performed while the cells were grown to 80% to 90% of the fullness of the dishes.

Preparation Example 4 Cell subculture

In the subculture of the cells, the culture dish containing the autologous umbilical cord original cells, which had been grown to 80% to 90% of the fullness of the culture dish, was taken out from the incubator and removed by a vacuum suction device. Culture medium. Next, each plate was washed with 5 ml of 1x PBS and the PBS was removed again. Thereafter, 3 ml of 0.25% trypsin was added to each plate of cells and placed in a 37 ° C incubator for 5 minutes. The attached cells were transferred to a 15 ml centrifuge tube and centrifuged at 400 g for 5 minutes. After removing the supernatant, 2 ml of the growth medium was added, and the cells were resuspended by aspirator. Next, 10 μl (μL) of the cell suspension and 10 μl of trypan blue dye were mixed, and the number of cells and the survival rate were counted by a cell counter. The number of cells at 2x10 ⁵ to 5x10 ⁵ and 6 ml of growth medium the cells were grown for further amplifying the number of cells in a petri dish. When cells are first subcultured, these cells are called primary cells, the P1 generation.

Example 1 Comparison of cell growth

Referring to Table 1, the human umbilical cord original cells to be subjected to proliferation culture prepared in Preparation Example 1 were cultured in the following two culture solutions: α-MEM culture solution was used as a culture solution, and 10 were added separately. % fetal bovine serum as the first group or 5% autologous umbilical cord plasma, 1ng/mL recombinant human basic fibroblast growth factor and 2U/mL heparin as the second group, the above groups are replaced every 3 days to 4 days 1 The secondary culture solution was changed 6 ml each time and cultured at 37 ° C under 5% carbon dioxide. When the number of the cell growth of about ⁷ to 10, a first set to 25 days to reach a number of 3.71x10 ⁷ cells, to reach a second set number of 6.14x10 ⁷ cells on day 24, even more than the first cells to a much group; when the number of cells to about 10 ^8, a first group 32 3.35x10 ⁸ days to reach the number of cells and the second group can be reached 9.62x10 ⁸ cells at day 31, It is also much more numerous than the first group. The effect of using autologous plasma on cell proliferation is shown to be better than the use of fetal bovine serum.

In addition, 10 ng/mL recombinant human basic fibroblast growth factor or 10 ng/mL recombinant human basic fibroblast growth factor and 2 U/mL heparin were added as a negative control with α-MEM culture medium as the culture solution. group. It was found that the cells were also cultured at 2×10 ⁵ cells. After the same number of days, the cells were labeled with trypsin and counted. The number of cells added with 10% fetal bovine serum was 3.3×10 ⁶ , but 10 ng/mL recombinant human alkaline was added. The number of cells of fibroblast growth factor decreased to 3x10 ⁴ , and the number of cells added with 10 ng/mL recombinant human basic fibroblast growth factor and 2 U/mL heparin also decreased to 7.5×10 ⁴ . Therefore, the addition of recombinant human basic fibroblast growth factor, or recombinant human basic fibroblast growth factor and heparin, does not affect the number of cells cultured in plasma- or serum-containing α-MEM culture medium.

Example 2 Comparison of cell growth after multiple generations Table 2. Number of cells after different generations of cells cultured with different additives.

Referring to FIG. 1 and Table 2, the human umbilical cord original cells to be subjected to proliferation culture prepared in Preparation Example 1 were cultured in the following two culture solutions: the α-MEM culture solution was used as the culture solution. Add 10% fetal bovine serum as the first group; 5% autologous umbilical cord plasma, 1 ng/mL recombinant human basic fibroblast growth factor and 2 U/mL heparin as the second group; 5% highly concentrated platelet lysate (available from Helios Bioscience, model UltraGro ^TM) as a third group; 10% concentrated platelet lysate (available from Helios Bioscience, model hPGF C18) as a fourth group; each group over 3-4 days every 3 days to replace The secondary culture solution was replaced with 6 ml each time, and the cultured cells were from P0 generation to P10 generation. As can be seen from Fig. 1, the number of cells cultured in the second group exceeded the first group from the P1 generation, and the number of cells cultured in the second group from the P7 generation began to significantly exceed the third group and the fourth group. The effect of using autologous plasma on cell proliferation is shown to be better than the use of fetal bovine serum, commercially available platelet lysate or the like.

The umbilical cord original cells of Example 1 and Example 2 were from different humans, and therefore there were some differences in the number of cells due to individual differences under the same treatment conditions, but it was found from the results that the second group added 5% autologous umbilical cord plasma, 1 ng/ The number of recombinant human basic fibroblast growth factor and 2 U/mL heparin in mL was better than that in the first group plus 10% fetal bovine serum.

It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, the various modifications that are apparent to those skilled in the art are also contemplated by the scope of the invention.

Claims (12)

A method for culturing progenitor cells in autologous plasma, comprising: preparing a primordial cell, wherein the primordial cell is a umbilical cord primitive cell; taking a culture solution; and adding a volume percentage of 5% autologous plasma Adding to the original cells and performing proliferation culture; wherein the autologous plasma system comprises the following steps: centrifuging the autologous blood twice; and filtering the filter to obtain the self for the original cell culture. Body plasma. The method according to claim 1, wherein the culture solution comprises minimum essential medium (MEM), α-MEM medium, basal media eagle (BME), and DMEM medium (Dulbecco's modified Eagle's modium, DMEM). MEM/F12 medium, Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium (RPMI), Medium 199 medium or a combination thereof. The method of claim 1, wherein the step of centrifuging the autologous blood twice, the first centrifugation speed is 400 g to 500 g, and centrifugation is performed for 5 minutes. The method of claim 1, wherein the step of centrifuging the autologous blood twice, the second centrifugation speed is from 1000 g to 1500 g, and centrifuging for 15 minutes. The method of claim 1, wherein in the step of filtering by a filter, the filter is a 0.22 μm filter. The method of claim 1, wherein the additive further comprises a growth factor. The method of claim 6, wherein the growth factor is recombinant human Basic fibroblast growth factor and concentration from 0.5 ng/mL to 10 ng/mL. The method of claim 7, wherein the concentration of the recombinant human basic fibroblast growth factor is 1 ng/mL. The method of claim 1, wherein the additive further comprises an anticoagulant. The method of claim 9, wherein the anticoagulant is heparin and the concentration is from 1 U/mL to 10 U/mL. The method of claim 10, wherein the concentration of the heparin is 2 U/mL. The method of any one of claims 1 to 11, wherein the autologous plasma is autologous umbilical cord plasma.