TWI454307B - Method and application for preparing human plasma for cell culture - Google Patents

Description

Method and application for preparing human plasma for cell culture

The present invention relates to a method of preparing human plasma for cell culture and isolating umbilical cord progenitor cells.

There are methods for culturing cells, and fetal bovine serum is most often used as a primary cell culture. With the development of the clinical application of cell therapy, the cells to be transplanted are safely used in patients, and the safety of cells cultured with fetal bovine serum is urgently considered.

Stem cells are a group of highly potential clinical application cell populations, so many research teams are investigating clinical trials. Hematopoietic stem cells (HSC) are commonly used for transplantation, while mesenchymal progenitor cells (MPC) are currently in clinical trials. Most of the stem cells used in clinical trials are cultured in medium containing fetal bovine serum. However, the use of fetal bovine serum may carry the risk of transmitting viral or pathogenic protein particles (prion), thus causing limitations in use.

In order to reduce the risk of human cell culture being infected by the aforementioned virus or pathogenic protein particles, if human cells can be cultured in human serum/plasma, the utilization, application and safety of human cells can be greatly improved. However, it is still unclear how to prepare human serum/plasma to replace the existing fetal bovine serum culture method. Therefore, there is an urgent need to propose a human serum/plasma for cell culture in order to safely use cell therapy and develop regenerative medicine.

On the other hand, most of the stem cells currently used in clinical practice are adult stem cells, such as bone marrow stem cells, and the placenta and umbilical cord system are medical wastes after maternal production, which contain many mesenchymal primitive cells. Therefore, if the primordial cells in the umbilical cord can be efficiently obtained in large quantities, it can also help the development of regenerative medicine.

In view of the deficiencies of the serum types for cell culture and the method for producing human serum/plasma for cell culture, the present invention is intended to provide a human plasma for cell culture. The present invention mainly utilizes medical waste: umbilical cord blood retained in the umbilical cord and the placenta, or general adult blood, to be separated and processed to obtain human plasma for cell culture.

On the other hand, the umbilical cord and placenta contain most undifferentiated stem cells, which can be effectively separated and preserved, which will greatly contribute to the progress of regenerative medicine. Accordingly, it is another object of the present invention to provide a method of separating protocells from the umbilical cord and the placenta.

In order to achieve the main object of the present invention, the present invention relates to a method for preparing human plasma for cell culture, which comprises: providing human blood to obtain plasma, and placing the obtained plasma into a dialysis tube with about 10 mM calcium chloride. Dialysis is performed twice, and human plasma for cell culture is obtained.

Preferably, the dialyzed plasma is further centrifuged at 3500 g and filtered through a 0.2 microporous filter paper to obtain human plasma for cell culture.

Another aspect of the invention relates to a method for isolating primordial cells from a umbilical cord, comprising: providing a umbilical cord; separating umbilical cord connective tissue from the umbilical cord and finely dividing the umbilical cord connective tissue; adding a decomposition enzyme to form a mixture for decomposition; Obtaining solids and liquids; and separately culturing solids and liquids to obtain umbilical cord original cells.

Preferably, the collected umbilical cord is first washed with alcohol, and then the umbilical vein and the umbilical artery are removed for nucleus connective tissue shredding.

Preferably, the aforementioned enzymes are trypsin and collagenase.

Preferably, the final concentration of trypsin is about 0.05% (v/v) and the final concentration of collagenase is about 300 U/ml.

Preferably, when the solid matter and the liquid material are separately cultured, α-MEM culture solution, about 20% (v/v) cord blood plasma and about 1% (v/v) antibiotic are added to each culture dish. Cultivate.

Another aspect of the invention relates to a human plasma for cell culture obtained by the aforementioned method.

By the method of the present invention, human serum/plasma can be applied to cells for clinical cell therapy or regenerative medicine, so as to effectively avoid the source of pollution from animals, and the method for collecting umbilical cord original cells provided by the present invention can be well. The original cells in the umbilical cord are collected to increase the utilization of stem cells.

The present invention relates to a method of preparing human plasma for cell culture and isolating primordial cells from the umbilical cord. In the present invention, the obtained human plasma is treated by dialysis using human blood to prepare a human plasma suitable for cell culture, and is used as a medium supplement for primary culture of in vitro stem cells. Since the preparation of the general serum is prepared by blood coagulation using blood without an anticoagulant; the plasma prepared by the present invention has been added with an anticoagulant due to the cord blood obtained at the beginning, thereby transmitting the present invention. The preferred dialysis method disclosed is, for example, dialysis with 10 mM CaCl ₂ to remove fibrinogen for culture of human cells.

The cell culture for human cells prepared by the method provided by the present invention can be used to culture cells for clinical trials, thereby avoiding the risk of animal-derived serum being infected by a contaminated source, thereby effectively ensuring the safety of clinical use or clinical trials. .

Additionally, the invention is further related to a method of separating protocells from an umbilical cord. Since the original cells can differentiate into different mother cells after appropriate external stimulation, the clinical application of more diverse stem cells can be provided by separating the original cells from the umbilical cord.

The main step of preparing the human plasma for cell culture comprises first providing human blood to obtain plasma, and arranging the obtained plasma into a dialysis tube for dialysis with about 10 mM calcium chloride, and then dialysis against physiological saline once. Finally, human plasma for cell culture is obtained.

The invention provides a method for separating primordial cells from an umbilical cord, which comprises the steps of: first collecting and collecting the umbilical cord, pulverizing the collected umbilical cord, adding a decomposition enzyme to the finely divided umbilical cord to form a mixture for decomposition, and then filtering the mixture to obtain Solids and liquids, and finally solids and liquids are separately cultured to obtain umbilical cord original cells. In a preferred embodiment of the invention, the degrading enzyme may be trypsin or collagenase.

The following terms are fully understood by those of ordinary skill in the art to which the invention pertains; the following definitions are intended to avoid any ambiguous explanation in the specific embodiments of the invention.

The term "human blood" as used in the present invention herein includes human blood or cord blood; preferably, cord blood.

The term "growth culture solution" as used in the present invention means herein that the α-MEM culture solution contains about 20% cord blood plasma and 1% antibiotic.

Other features and advantages of the present invention will be apparent from the following detailed description of the invention.

Example Example 1: Preparation of cord blood plasma for cell culture

The fresh cord blood plasma obtained by separating the cord blood to which the anticoagulant has been added is stored at 4 ° C. The blood cell separation technique is generally known in the art and can be carried out according to the prior art before the application. Approximately 20 ml of cord blood plasma was placed in a dialysis tube (3500 MWCO), dialyzed against 1 liter of 10 mM CaCl ₂ at room temperature, and dialyzed overnight at 4 ° C for dialysis. The dialysis was then carried out in 500 ml of physiological saline at room temperature for a dialysis equilibrium time of about 4 hours (or overnight dialysis at 4 ° C).

Blood clot production can be observed in the dialysis tube. The contents of the dialysis tube were then transferred to a 50 ml centrifuge tube and centrifuged at 3500 G for 15 minutes to remove blood clots, and the resulting supernatant was cord blood plasma for cell culture. The supernatant was then filtered through a 0.2 microporous filter paper, and the filtered cord blood plasma was collected and stored at 4 °C.

Example 2: Isolation of umbilical cord progenitor cel

The collected umbilical cords were washed and disinfected with 75% alcohol and then washed with PBS. The umbilical cord was cut into 4 to 6 fragments, and each fragment was placed on a petri dish. Insert the umbilical cord with sharp scissors and cut the veins, and spread the umbilical cord with tweezers. The two umbilical arteries were separated by forceps and carefully peeled off to avoid any blood splashing on the umbilical cord connective tissue while removing the umbilical vein. The umbilical cord connective tissue was placed on a small amount of a-MEM to keep it moist. The umbilical cord connective tissue is then cut into finely divided pieces with surgical scissors. The chopped umbilical cord connective tissue is divided into labeled centrifuge tubes to give an amount of about 10 ml (ml) per tube, and 4 ml (ml) of about 0.25% trypsin and 1 ml (ml) are added. The 300 U/ml collagenase solution gave a final concentration of trypsin of about 0.05% (v/v), a final concentration of collagenase of about 300 U/ml, and a volume of each tube of 20 ml of the mixture.

The tubes containing the chopped umbilical cord connective tissue were cultured in a carbon dioxide incubator at 37 ° C for one hour, after which the mixture was filtered through a sterile mesh to separate solids and liquids, and the filtered liquid was collected into 50 ml test tubes. in. The filtered solid portion was cultured on a culture dish with 10 ml (ml) of 20% cord blood plasma culture medium, and an additional 1 ml of cord blood plasma was added, which is called tissue separation culture. (diffusion culture).

After the filtered liquid was centrifuged at 400 G for 10 minutes, the supernatant was removed to 5 ml, and the suspension was again suspended in the suspension state. Further, 10 ml of a 20% cord blood plasma culture solution was added, and 15 ml of the growth medium containing the cells was seeded in a 10 cm culture dish, and each culture dish was cultured in a carbon dioxide incubator at 37 °C. On the next day, confirm whether the Petri dish is contaminated under a microscope. If there is no contamination, discard the old culture solution and wash it twice with 5 ml of 1X PBS. The foregoing operation was carried out using Pasteur pipettes. Add 6 ml of 20% cord blood plasma culture medium to the culture dish, and place the culture dish again in a carbon dioxide incubator for cultivation. The culture medium was changed twice a week, and each time it was replaced, it was observed under a microscope, and after washing once with 5 ml of PBS, 6 ml of fresh medium was added.

Further, after about 3-4 days of the solid portion cultured by the tissue separation method, the solid portion and the culture solution are removed, and the culture solution is washed and renewed in the aforementioned manner. Umbilical cord primitive cells are obtained from the filtrate and solid fraction.

The umbilical cord primitive cell obtained by the foregoing method is as shown in the first figure, and forms a fibroblast-like appearance when the culture is late. At the same time, please refer to the second and third figures respectively, and further analyze the obtained cell surface antigen by flow cytometry, and find that the obtained cell surface carries CD29, CD44, CD73, CD90, CD105, CD166 and HLA-ABC. Antigen labeled without CD14, CD34, CD45 and HLA-DR surface antigens.

Example 3: Subculture of umbilical cord primitive cells

After about 10-15 days, when the colonies grow, the cells can be subcultured. First, prepare a 15 ml centrifuge tube and 0.25% trypsin-EDTA (trypsin-EDTA). The culture dish was removed from the incubator, and the culture solution was removed, and then washed once with 5 ml of PBS. After adding 3 ml of trypsin-EDTA to each dish, it was placed in a 37 ° C incubator for 5 minutes. The cells separated by the culture dish were transferred to a 15 ml centrifuge tube and centrifuged at 400 G for 5 minutes to collect the cells. The supernatant after centrifugation was removed and the cells were resuspended in 1-2 ml of growth medium. Ten microliters (μl) of the cell suspension was mixed with 10 microliters (μl) of trypan blue, and then counted by a hemocytometer (dilution factor of 2).

The cells isolated as described above were seeded at a concentration of 3-5×10 ⁵ cells per dish, and 20% cord blood plasma culture solution was added to each dish to 6 ml, and then placed at 37 ° C. The carbon dioxide incubator is cultured. These cells are called primary cells, the P1 generation.

The culture solution was updated every 3-4 days. First, the cells should be observed under a microscope. If they grow to a growth density of 90%, the cells are further subcultured. The culture dish was taken out from the incubator, and the culture solution was removed and washed twice with 5 ml of PBS. After adding 3 ml of trypsin-EDTA to each culture dish, the culture dish was placed in a 37 ° C incubator for 5 minutes, after which the cells separated from the culture dish were transferred to a 15 ml centrifuge tube and centrifuged at 400 G. Minutes to collect cells. A portion of the supernatant was removed and 2 ml of the supernatant was left to resuspend the cells. Ten microliters (μl) of the cell suspension was mixed with 10 μl of trypan blue, and the number of cells (dilution factor 2) was calculated by a hemocytometer. The above cell suspension was contained in each culture dish containing 3-5×10 ⁵ cells, and evenly implanted into each culture dish, and 20% cord blood plasma culture solution was added to each culture dish to 6 ml. The cells were cultured in a carbon dioxide incubator at 37 °C. The medium was changed every 3-4 days. The cells were observed under a microscope before each change. When the cells were grown to a plate area of about 90%, the cells were subjected to FACS analysis and cryopreserved.

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.

The first image is a photographic view of the culture state of mesenchymal cells.

The second panel is a flow cytometric result plot of the mesenchymal blast cells with CD44, CD73 and CD105 antigen markers.

The third panel is a flow cytometry result plot of mesenchymal cells with CD29 and HLA-ABC antigen markers without CD34 antigen labeling.

Claims (7)

A method for preparing human plasma for progenitor cell culture in vitro comprises: placing a plasma into a dialysis tube for dialysis once with about 5-20 mM calcium chloride, and then dialyzing with an isotonic solution once, The dialyzed plasma was further centrifuged at 3500 g, the supernatant was taken, and the precipitate was discarded, and the supernatant was filtered through a 0.2 (μm) microporous filter paper; human plasma for the original cell culture was obtained. The method of claim 1, wherein the equilibrium time for dialysis is about 4 ° C for 12-16 hours or room temperature for 4-6 hours. The method of claim 2, wherein the plasma is dialyzed against about 10 mM calcium chloride. The method of claim 3, wherein the isotonic solution is physiological saline. A method for applying human plasma obtained in the first to fourth aspects of the patent application to culture umbilical cord original cells, comprising: α-MEM culture solution, about 15-30% (v/v) human plasma, about 1 % (v/v) antibiotics were cultured. The method of claim 5, wherein the human plasma system is about 20% (v/v). A human plasma for umbilical cord primitive cell culture obtained by the method of claims 1 to 4 of the patent application.