Serum substitute and its preparation method and use

Abstract

The present invention provides a serum substitute and its preparation method and use, the serum substitute comprising fetal bovine or newborn bovine-derived platelet lysate and plasma, and can be prepared by dissolving fetal bovine or newborn bovine-derived platelet-rich plasma.

Description

[Technical field]

[0001] The present invention relates to a serum replacement and its preparation method and use. [Background technology]

[0002] Stem cells are a type of pluripotent cell with the ability to self-renew and, under certain conditions, can differentiate into various functional cells. Mesenchymal stem cells are one of the members of the stem cell family and are present in various tissues such as hair follicles, umbilical cord, uterine membrane, and fat, and have the ability to differentiate in multiple directions. These stem cells have the ability to differentiate into three lineages of mesenchymal stem cells, such as osteoblasts, chondrocytes, and adipocytes, as well as the ability to differentiate into other cell types, and also have unique cytokine secretion control functions, immune control functions, and anti-inflammatory functions.

[0003] Blood is composed of a liquid component, plasma, and blood cells suspended in it, collectively called whole blood. After blood separated from the body is anticoagulated, it is centrifuged and precipitated, and the liquid without cellular components is called plasma. Plasma can be considered as the extracellular matrix of blood, and its main function is to carry blood cells and transport substances necessary to maintain vital activities and waste products produced in the body. After blood separated from the body is coagulated, the liquid released by the clotting of the clot is serum, in which fibrinogen and certain clotting factors in the plasma are removed; its main function is to provide basic nutrients, hormones, and various growth factors. In general, the main difference between plasma and serum is that plasma contains fibrinogen. Platelets are one of the main forming components of blood and play an important role in the process of hemostasis. In addition, during the process of platelet activation and degranulation, a large number of factors involved in inflammatory responses and tissue repair are released.

[0004] Highly efficient cultivation of stem cells has always been a challenge that the field hopes to solve, and culture medium is an essential part of the cell culture process. Animal serum, as one of the main components of stem cell culture medium, plays an important role in cell growth and regeneration. Among animal serum, bovine serum is the most widely used one, and therefore bovine serum is one of the important raw materials for biopharmaceuticals. According to the time of blood collection, bovine serum can be divided into fetal bovine serum, new born calf serum, calf serum, and adult bovine serum. However, animal serum is not only expensive, but also has uncontrollable quality and large stability differences from batch to batch. Therefore, more and more researchers have begun to develop serum substitutes, for example, patent publication number CN107384856A discloses a method for preparing platelet lysate, and patent publication number CN101486996A discloses a non-animal derived cell culture serum substitute. All known serum substitutes are human-derived and available in small quantities, therefore there is an urgent need to develop serum substitutes with broad source availability, high yield, safety, and stability. Summary of the Invention [Means for solving the problem]

[0005] Contents of the invention The present invention uses a controllable bovine blood source, ensures its traceability, and implements strict quality control standards. In addition, by adopting the serum replacement manufacturing method provided by the present invention, a high-quality, safe and stable serum replacement product can be obtained. The serum replacement product of the present invention meets the quality control standards for newborn bovine serum in the General Chapter of the Pharmacopoeia of the People's Republic of China (2020 edition).

[0006] In one aspect, the present invention provides a serum replacement comprising platelet lysate and plasma, the platelet lysate and plasma being derived from fetal bovine or newborn bovine. In some embodiments, the serum replacement is comprised of fetal bovine or newborn bovine platelet lysate and plasma. Preferably, the serum replacement is prepared by the method for preparing a serum replacement of the present invention.

[0007] In another aspect, the present invention provides a method for preparing a serum replacement, said method comprising platelet lysing platelet-rich plasma from fetal or newborn bovine calf.

[0008] In some embodiments, the method further comprises separating and removing blood cells other than platelets from fetal bovine whole blood or newborn bovine whole blood to obtain the platelet-rich plasma; and, optionally, comprising a sterile grade filtration step after lysing the platelets.

[0009] In some embodiments, blood cells other than platelets are separated and removed by centrifugation, preferably differential centrifugation, preferably under low temperature conditions.

[0010] In some embodiments, platelet lysis employs a freeze-thaw method.

[0011] The present invention further provides the use of the serum substitute of the present invention in culturing stem cells and in inducing stem cell differentiation. The stem cells include, for example, mesenchymal stem cells, such as human hair follicle mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells, and human uterine mesenchymal stem cells. The serum substitute of the present invention can significantly improve the proliferation and differentiation ability of stem cells such as mesenchymal stem cells compared to existing animal sera. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the cell yields obtained by culturing primary umbilical cord stromal stem cells separately in medium containing fetal bovine serum and in medium containing a fetal bovine serum substitute of the present invention. [Diagram 2]FIG. 2 shows the cell yields obtained by culturing primary hair follicle stromal stem cells separately in a medium containing fetal bovine serum and a medium containing a fetal bovine serum substitute of the present invention. [Diagram 3] FIG. 3 shows adipogenic differentiation of hair follicle stromal stem cells induced in medium containing fetal bovine serum and medium containing a fetal bovine serum substitute of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Unless otherwise specified, the technical features described in this specification, such as the contents, steps, condition parameters, etc., are not limited to the combinations of the embodiments and examples specifically described in this specification, and any other combinations thereof also fall within the scope of the present invention.

[0014] In this specification, unless otherwise specified, numerical ranges expressed by endpoints are considered to specifically disclose every number between those endpoints, and any combination of any two of those numerical ranges. Furthermore, unless otherwise specified, when any numerical ranges or values ​​for the same parameter are specifically recited, those endpoints and values ​​can be combined in any way, and the resulting range, if included in the largest continuous range specifically recited, is within the scope of the invention.

[0015] Herein, unless otherwise specified, the numerical values ​​or numerical ranges, whether preceded by the word "about" or not, cover an approximate range equivalent to the numerical value or numerical range that would be understood by one of ordinary skill in the art, for example, a range of ±10% of the numerical value or end value, and may be a range of ±5%, ±3%, ±2%, ±1% or ±0.5%.

[0016] Unless otherwise specified herein, the meanings of all scientific and technical terms are consistent with the meanings known or known in the art, particularly in the stem cell, mesenchymal stem cell, and culture media fields, e.g., as described in textbooks, test handbooks, or existing technical literature.

[0017] Here, an open phrase such as "comprising" means that it does not exclude unspecified elements, such as other ingredients in the composition or other steps in the method; a closed phrase such as "consisting of" includes only the elements specified, but may include impurities present in normal amounts in the composition, and may include steps in the method that are not specified but that occur substantially inevitably. Open phrases are considered to cover and specifically disclose the situation limited by the closed phrase.

[0018] Here, features numbered with numbers or letters, e.g., steps, such as "a", "b", ..., "I", "II", ..., "first (1)", "second (2)", etc., have no sorting meaning, unless otherwise specified, but only a distinguishing or counting meaning.

[0019] Herein, unless otherwise indicated, the conjunction "or" is synonymous with "and / or" and means that one or more items of parallel items or circumstances are permitted to be included, unless otherwise stated, and unless there are incompatible elements. For example, in this context, "derived from bovine fetus or newborn bovine" indicates that it may be derived from bovine fetus or newborn bovine, and includes the simultaneous presence of both origins.

[0020] The present invention provides a serum replacement, which comprises platelet lysate and plasma derived from fetal or newborn bovine calves.

[0021] In this specification, the identification of a bovine fetus or a newborn cow is well known to those skilled in the art and is usually defined according to the age of the cow and whether it has eaten or not. For example, a bovine fetus is a bovine embryo of a pregnant cow that is 5 to 8 months old, and a newborn cow may be a calf that is not fed and is within 14 hours of birth. A calf may be a cow that is within 1 year of age, and an adult cow may be a cow that is 1 year of age or older.

[0022] In some embodiments, the serum replacement consists of platelet lysate and plasma, but does not eliminate impurities that are normally present in content.

[0023] Preferably, the serum replacement can be prepared by the method for preparing a serum replacement of the present invention described below, i.e., the serum replacement can be obtained by dissolving platelets from platelet-rich plasma derived from fetal bovine or newborn bovine.

[0024] The present invention also provides a method for preparing a serum substitute and a serum substitute prepared thereby, the method comprising platelet dissolving platelet-rich plasma derived from fetal bovine or newborn bovine.

[0025] In the present invention, "platelet-rich plasma" refers to plasma that contains platelets but does not contain other blood cells such as red blood cells and white blood cells, and includes, for example, platelet-rich plasma, concentrated platelets, etc. For example, platelet-rich plasma can be produced by separating and removing blood cells other than platelets, such as red blood cells and white blood cells, from whole blood.

[0026] In the present invention, whole blood for producing platelet-rich plasma can be preferably collected from fetal bovine or newborn bovine. Before collecting bovine blood, a common anticoagulant is added to the collection container to prevent the bovine blood from clotting. Anticoagulants commonly used in the art include oxalates, heparin, citrates, ethylenediaminetetraacetic acid (EDTA), etc. In a preferred embodiment, the anticoagulant is sodium heparin. When collecting bovine blood, it should be noted that the collection site needs to be disinfected, and the syringe and test tube used for blood collection should be clean and dry.

[0027] In one embodiment, the platelet-rich plasma is produced by utilizing the density difference between blood cells and plasma, and then obtaining the target component by using a centrifugal technique to separate different components. In the present invention, when the platelet-rich plasma is obtained by centrifuging, the supernatant after centrifugation is collected to obtain the platelet-rich plasma.

[0028] Common centrifugation techniques include sedimentation centrifugation, differential centrifugation, and density centrifugation. Differential centrifugation involves increasing the centrifugation speed stepwise or alternating high-speed and low-speed centrifugation to separate and settle substances with different sedimentation coefficients from the mixture to the bottom of the tube. In some embodiments, multiple centrifugations may be performed to improve the yield, and differential centrifugation with different rotation speeds is preferred. After one or more low-speed centrifugations, the remaining portion from which the supernatant is removed is subjected to one or more high-speed centrifugations, and the collected supernatants are combined. Preferably, blood cells other than platelets (hereinafter abbreviated as "other blood cells") can be separated and removed by the differential centrifugation method.

[0029] Preferably, to avoid inactivation of the sample during the preparation process, low-temperature centrifugation (e.g., low-temperature differential centrifugation) is used to separate and remove other blood cells. For example, the low temperature may be 10°C or lower, or 5°C or lower, e.g., 4°C or lower.

[0030] Centrifugal speed can be expressed in different units. The rotation speed during low-speed centrifugation is usually expressed in revolutions per minute (rpm), while the rotation speed during high-speed centrifugation is expressed in "g" or "xg". However, "g" and "xg" both represent multiples of the gravitational acceleration, and the two are synonymous and can be replaced with each other, for example, 1000g and 1000xg are synonymous. Relative centrifugal force represents the ratio of centrifugal force to gravity, and is equal to the number before "g" or "xg", for example, a centrifugal force of 1000g or 1000xg, i.e., the relative centrifugal force is 1000.

[0031] In the present invention, other blood cells can be removed using one or more centrifugations at the same or different centrifugation speeds, which may be 400g to 2100g, for example 800g to 2000g, 400g to 1000g, or 1800g to 2100g, and the centrifugation time may be 10 to 30 minutes, for example 15 to 20 minutes, 10 to 25 minutes, or 15 to 30 minutes. In some embodiments, the removal of other blood cells by centrifugation includes at least one centrifugation at 400g to 1000g for a time of 10 to 25 minutes, and is preferably performed at a low temperature, for example 4°C.

[0032] In some embodiments, differential centrifugation is used to remove other blood cells. In some embodiments, the differential centrifugation includes at least one low-speed centrifugation followed by at least one high-speed centrifugation. In such an embodiment, a relatively low-speed centrifugation condition is set to preliminarily separate blood cells and plasma, and the platelets to be retained are dissolved in the plasma to obtain a supernatant layer of platelet-containing plasma with retained platelets and a precipitate layer rich in other blood cells. Any centrifugation condition that can obtain such an effect can be the condition of "low-speed centrifugation" of the present invention. For example, the low-speed centrifugation can be performed at a rotation speed of 400g to 1000g for a time of 10 to 25 minutes, preferably at a low temperature, for example, 4°C. For example, the rotation speed of the centrifugation can be 400g, 600g, 800g, or 1000g; the time can be 10 minutes, 15 minutes, 20 minutes, or 25 minutes. In one preferred embodiment, the low-speed centrifugation is performed at a rotation speed of 400 g for 15 minutes at a temperature of 4° C. In another preferred embodiment, the low-speed centrifugation is performed at a rotation speed of 800 g for 10 minutes at a temperature of 4° C. If not used immediately, the supernatants obtained after each low-speed centrifugation may be frozen or stored together, for example at −80° C. to −4° C.

[0033] In this embodiment, a relatively high-speed centrifugation condition is set, and the remaining portion after removing the supernatant after the low-speed centrifugation is centrifuged again to perform secondary separation of the plasma and blood cells that were not completely separated. Any centrifugation condition that can achieve this effect can be considered as the "high-speed centrifugation" condition of the present invention. In this way, completely separated plasma can be obtained. Normally, completely separated plasma is thought to be free of platelets.

[0034] As used herein, the expression "free of" a certain substance includes a situation in which the substance is substantially free, i.e., the substance is below the detection limit, below the standard, and / or present in only minute amounts that do not affect the intended use and intended effect of the product in which the substance is present (e.g., the serum substitute of the present invention), as will be understood by those skilled in the art.

[0035] For example, the rotation speed of the high-speed centrifugation may be 1800g to 2100g, the time may be 15 to 30 minutes, and it is preferable to perform the centrifugation at a low temperature, for example, 4°C. For example, the rotation speed of the centrifugation may be 1,800g, 1,900g, 2,000g, or 2,100g; the time may be 15 minutes, 20 minutes, 25 minutes, or 30 minutes. In one preferred embodiment, the rotation speed of the high-speed centrifugation is 2,000g, the time is 20 minutes, and the temperature is 4°C; in another preferred embodiment, the rotation speed of the high-speed centrifugation is 2,100g, the time is 15 minutes, and the temperature is 4°C. If not used immediately, the supernatants obtained by separation from each high-speed centrifugation may be frozen and stored separately or together, for example, frozen and stored at -80°C to -4°C.

[0036] For dissolving platelets, a method of repeating thawing and freezing (freeze-thaw) can be adopted. The freeze-thaw method can completely rupture platelets dissolved in plasma and allow their contents to flow out. There is no limit to the number of freeze-thaw cycles, as long as the above-mentioned effect of complete rupture can be obtained. The number of freeze-thaw cycles may be counted from the time when a frozen-preserved sample is re-frozen after the first thawing, or may be counted from the time when a non-frozen-preserved sample is re-thawed after the first freezing. In one embodiment of the present invention, the number of freeze-thaw cycles is 3-7, for example, 3, 4, 5, 6, or 7 times, and in one preferred embodiment, it is 6 times, and in another preferred embodiment, it is 4 times.

[0037] During freezing, it is necessary to ensure that the clear liquid is completely frozen, and the freezing time and temperature vary according to actual conditions. Preferably, the freezing time is 8 hours or more. For example, the freezing time can be 8 hours, 9 hours, 10 hours, 12 hours, or 15 hours. Preferably, the freezing temperature is below -20°C. For example, the freezing temperature can be -20°C, -25°C, -40°C, -45°C, -60°C, -80°C, -100°C, or -150°C. In one preferred embodiment, the freezing temperature is -80°C, and in another preferred embodiment, it is -45°C.

[0038] Preferably, when melting, the liquid in the bottle is in the form of an ice-water mixture, for example forming ice cubes the size of a fingernail, and does not increase in temperature. In a preferred embodiment, the temperature of the melt is 0°C.

[0039] In the present invention, the purpose of repeating the freezing and thawing is to break down platelets, and the supernatant obtained by low-speed centrifugation is rich in platelets. Therefore, in a preferred embodiment, the supernatant obtained by low-speed centrifugation is mixed with the supernatant obtained by high-speed centrifugation after repeated freezing and thawing, and the subsequent operations are carried out. To simplify the operations, the supernatants obtained by low-speed centrifugation and high-speed centrifugation can also be mixed to carry out the subsequent operations. Therefore, in another preferred embodiment, the supernatants obtained by low-speed centrifugation and high-speed centrifugation are mixed, and then the freezing and thawing are repeated.

[0040] In the present invention, a sterilization grade filtration step may be included after repeated freezing and thawing. Sterilization grade filtration refers to filtration that provides a sterilization effect, and the intended and / or actual effect is not necessarily limited to sterilization, but also includes, for example, removing other impurities and microorganisms such as cell debris, mycoplasma, etc. In one preferred embodiment, filtration is performed using a 0.22 μm filter membrane. In another preferred embodiment, filtration with a 0.22 μm filter membrane can be performed again with a 0.1 μm filter membrane. In addition to sterilization, the 0.1 μm filter membrane can further remove mycoplasma, increase the safety of the serum replacement, and do not affect the performance (e.g., proliferation ability) of the stem cells obtained by culture.

[0041] The present invention further provides a use of the serum replacement of the present invention in culturing stem cells. The present invention further provides a use of the serum replacement of the present invention in inducing stem cell differentiation. Stem cells include embryonic stem cells, adult stem cells, (induced) pluripotent stem cells, etc. Pluripotent stem cells specifically include mesenchymal stem cells, such as human hair follicle mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells, and human uterine mesenchymal stem cells. The adult stem cells may be adipose stem cells, uterine mesenchymal stem cells, hair follicle stem cells, or umbilical cord stem cells. It should be noted that the term "stem cells" in the present invention does not include cells isolated or obtained from human embryos that are more than 14 days old after fertilization, or cells that have developed in vivo. In some embodiments, the induction of stem cell differentiation may be, for example, inducing adipogenic differentiation of stem cells or inducing chondrogenic differentiation of stem cells. The serum replacement has shown good effects in inducing adipogenic or chondrogenic differentiation of stem cells. In some embodiments, the content (volume ratio) of the serum replacement in the medium is 5% to 15%. Preferably, the content (volume ratio) of the serum replacement in the medium is 10%.

[0042] In a preferred embodiment, the stem cells include mesenchymal stem cells, such as human hair follicle mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells and human uterine mesenchymal stem cells. EXAMPLES

[0043] The present invention will be described in more detail below with reference to examples. However, it should be understood that these examples are for illustrative purposes only and do not limit the scope of the present invention.

[0044] In this specification and drawings, "blood substitute" is an abbreviation for serum substitute.

[0045] Example 1 Preparation of serum replacement In this embodiment, fetal bovine blood, newborn bovine blood, calf blood and adult bovine blood are used to prepare fetal bovine serum substitute, newborn bovine serum substitute, calf serum substitute and adult bovine serum substitute. Fetal bovine blood is bovine blood collected by puncturing the heart of a fetal bovine in a cow that is 8 months pregnant; newborn bovine blood is collected from a newborn cow that is not eating less than 14 hours after birth; calf blood is collected from a 6-month-old calf; adult bovine blood is collected from an 18-month-old adult cow. The source of bovine blood is traceable and strict quality control indicators are implemented.

[0046] Collection and transportation of bovine blood Before collecting the bovine blood, 100 mg of heparin sodium was added to a 500 mL serum bottle so that the final concentration of heparin sodium was 200 mg / L; during the process of collecting the bovine blood, the serum bottle was gently shaken to mix the bovine blood and heparin sodium uniformly; after collection, the bovine blood was transported to the serum substitute preparation laboratory at 4°C.

[0047] Preparation of serum replacement The collected bovine blood was placed in a biological safety cabinet and distributed; the first low-speed centrifugation: centrifuged at 400g for 15 minutes at 4°C, and the centrifuge tube was slowly removed from the centrifuge, at which point it was separated into an upper supernatant and a lower sediment. The lower sediment consisted of a white blood cell layer close to the top and a red blood cell layer at the bottom. The upper supernatant was collected in a 250mL serum bottle, labeled A1, and frozen at -80°C; the second high-speed centrifugation: the lower sediment after removing the upper supernatant was centrifuged at 2000g for 20 minutes at 4°C, and the upper supernatant was carefully aspirated and placed in a 500mL serum bottle, labeled A2, and frozen at -20°C.

[0048] A1 was taken out and equilibrated at room temperature for 10 minutes, then placed in a 37°C constant temperature water bath, and the bottle was immersed in water in the water bath by more than two-thirds of the bottle, and thawed by shaking at a speed of 80 to 100 revolutions per minute. Observations were made every 10 minutes, and when the state of ice-water mixture in the bottle (0°C) was reached, the bottle was immediately taken out and thawing was terminated; A1 was again frozen and stored in a -80°C ultra-low temperature refrigerator for more than 8 hours until it was completely frozen, which is recorded as one cycle of freezing and thawing; freezing and thawing (i.e., thawing and freezing) were repeated six times in this manner.

[0049] After six freeze-thaw cycles, A1 and A2 were thawed in a 37°C constant temperature water bath, respectively, and then A1 and A2 were mixed homogeneously in a biological safety cabinet; finally, after filtering through a 0.22 μm filter membrane, serum substitute (abbreviated as "blood substitute") was obtained; this was distributed into new serum bottles and stored frozen at a temperature of -15°C or below.

[0050] Example 2 Cultivation of human hair follicle stromal stem cells with serum replacements of different origins Obtaining primary human hair follicle stromal stem cells Intact human hair follicle tissues were obtained and placed in 10 cm petri dishes containing 10 mL of medium containing bovine blood replacement, which was DMEM / F12 medium (purchased from Shanghai Yuanpei Biological Biotechnology Co., Ltd.) supplemented with 1% (v / v) penicillin / streptomycin (purchased from Biyuntian Biotechnology Institute) + 10% fetal bovine blood replacement, newborn bovine blood replacement, calf blood replacement, or adult bovine blood replacement, respectively. However, fetal bovine blood replacement, newborn bovine blood replacement, calf blood replacement, and adult bovine blood replacement were prepared by the method described in Example 1. Using a microscope, the human hair follicle tissue was carefully placed at the bottom of a 1.5 mL EP tube, and a pipette was used to aspirate as much of the medium contained in the human hair follicle tissue as possible, and 8 μL of enzyme digestion solution was added to each hair follicle. The EP tube containing the enzyme digested cell suspension was placed in a constant temperature metal bath at 37°C and enzyme digested for 3 hours, with the rotation speed set to 600 rpm (revolutions per minute).

[0051] The employed enzymatic digestion solution was prepared by mixing the following components: 0.05 mL 20 mg / mL collagenase IV (Sigma-Aldrich), 0.05 mL 40 mg / mL Dispase II (Sigma-Aldrich), 0.01 mL 500 mM CaCl2 (Sigma-Aldrich), and 0.89 mL HBSS (balanced salt solution, Shanghai Yuanpei Biotechnology Co., Ltd.).

[0052] After the enzymatic digestion was completed, it was found under the microscope that the outer root sheath of the hair root was completely enzymatically digested, while the hair shaft portion could not be completely enzymatically digested. The EP tube was gently sprayed 20 times with a 20-200μL pipette to thoroughly mix the enzymatic digestion solution. After leaving it for 1 minute, the unenzymatically digested hair shaft sank to the bottom of the EP tube, and the upper layer of the enzymatic digestion suspension was aspirated, which is the primary (P0 generation) interstitial stem cells. The same amount of AO / PI reagent was added, mixed uniformly, and counted using a Countstar fluorescent counter. For details on the use of the automatic cell counter, please refer to the "Countstar Rigel S2 Type Automatic Cell Counter Standard Operating Instructions" WWSC-0024-CC.

[0053] Obtaining first generation hair follicle stromal stem cells 2 mL of the DMEM / F12 medium containing 10% fetal bovine blood replacement, newborn bovine blood replacement, calf blood replacement, or adult bovine blood replacement + 1% penicillin / streptomycin was taken out and placed in the bottom of each centrifuge tube in advance. A cell sieve was placed on top of the wells of a six-well plate, and 40 μL of the enzyme-digested cell suspension, corresponding to 5 hair follicles per well, was taken out and passed through a 100 μm cell sieve. Then, the medium containing various bovine blood replacements prepared in advance in the centrifuge tube was taken with the original pipette tip and passed through the cell sieve, and this was repeated three times. Using a 20-200μL pipette, the culture medium containing various types of bovine blood substitutes prepared in advance in centrifuge tubes of 200μL or more was aspirated, the EP tube was washed, and then the medium was passed through a cell sieve three times. Finally, the remaining culture medium containing various types of bovine blood substitutes prepared in advance in the centrifuge tubes was passed through a cell sieve. The six-well plate was incubated in a 37℃ 5% CO2 cell incubator. After static culture for 5 days, the cells were photographed, observed, and the liquid was replaced. The liquid was replaced on the 5th and 7th days.

[0054] After culturing for 10 to 12 days, when the cell density in the six-well plate reached 80% or more, the medium was discarded, 0.5 mL of TrypLE was added to the six-well plate, and digested at room temperature for 3 minutes. Using a pipette, 0.5 mL of the above-mentioned 10% fetal bovine blood replacement, newborn bovine blood replacement, calf blood replacement, or adult bovine blood replacement + 1% penicillin / streptomycin-containing DMEM / F12 medium was added to each culture bottle correspondingly to stop the digestion, and after inoculation, first generation (P1 generation) human hair follicle stromal stem cells were obtained.

[0055] Add an equal volume of AO / PI reagent, mix uniformly, and count using a Countstar fluorescence counter. For details on the use of the automated cell counter, refer to the "Countstar Rigel S2 Automated Cell Counter Standard Operating Instructions" WWSC-0024-CC. Here, the amplification factor = number of passage harvested cells / number of inoculations. In this example, the amplification factor was strictly 5000 cells / cm. 2 The area of ​​one six-well plate was 10 cm. 2i.e., 0.5 x 10 cells per six-well plate each time. 5 When cells are inoculated, the formula for calculating the amplification fold is "Amplification fold = number of cells harvested each time / (0.5 x 10 5 )".

[0056] According to the above method, hair follicle tissues from two different donors were harvested, and the cell yield and amplification fold obtained at the P0 and P1 generations are shown in Table 1 below. [Table 1]

[0057] From the above, it was shown that when used in culture medium, fetal bovine blood substitute and newborn bovine blood substitute can significantly enhance the proliferation ability of primary and subcultured interstitial stem cells, and that fetal bovine blood substitute is slightly superior to newborn bovine blood substitute.

[0058] Example 3 Preparation of serum replacement In this embodiment, the bovine blood described is bovine fetal blood, which is obtained by puncturing the heart of a bovine fetus in the body of a female cow at 8 months of pregnancy.

[0059] Collection and transportation of bovine blood Before collecting the bovine blood, 100 mg of heparin sodium was added to a 500 mL serum bottle so that the final concentration of heparin sodium was 200 mg / L; during the process of collecting the bovine blood, the serum bottle was gently shaken to mix the bovine blood and heparin sodium uniformly; after collection, the bovine blood was transported to the serum substitute preparation laboratory at 4°C.

[0060] Preparation of serum replacement The collected bovine blood was placed in a biological safety cabinet and distributed; the first low-speed centrifugation: centrifuged at 800g for 15 minutes at 4℃, and the centrifuge tube was slowly removed from the centrifuge, at which point it was separated into an upper layer of supernatant and a lower layer of sediment. The lower layer of sediment consisted of the white blood cell layer near the top and the red blood cell layer at the bottom. The upper layer of supernatant was collected in a 250mL serum bottle, labeled A1, and stored at 4℃; the second high-speed centrifugation: the lower layer of sediment after removing the upper layer of supernatant was centrifuged at 2100g for 15 minutes at 4℃, and the upper layer of supernatant was carefully aspirated and placed in a 500mL serum bottle, labeled A2, and A1 and A2 were mixed uniformly and labeled A3. A3 was frozen and stored at -80℃.

[0061] A3 was taken out and equilibrated at room temperature for 10 minutes, then placed in a 37°C constant temperature water bath, and thawed by immersing more than two-thirds of the bottle in the water bath and shaking at a speed of 80-100 rpm. Observations were made every 10 minutes, and when the temperature inside the bottle became a mixture of ice and water (0°C), the bottle was immediately taken out and the thawing was completed; A3 was again frozen and stored in a -45°C ultra-low temperature refrigerator for more than 12 hours until it was completely frozen, which is recorded as one cycle of freezing and thawing; this freezing and thawing process (i.e., thawing and freezing) was repeated four times.

[0062] After four freeze-thaw cycles, A3 was thawed in a constant temperature water bath at 37°C. Finally, after filtration through a 0.22 μm filter membrane, serum substitute ("blood substitute") was obtained; it was dispensed into new serum bottles and stored frozen at a temperature of -15°C or lower.

[0063] Example 4 Comparison of serum and serum replacements in primary human mesenchymal stem cell cultures Comparison of serum and serum substitutes in the culture of primary human umbilical cord stromal stem cells Intact human umbilical cord tissue was obtained and cut 1 cm from each end of the cord with large surgical scissors, followed by multiple rinses to remove blood, and then the outer amniotic layer was cut away along the umbilical vein only with small scissors, bluntly separated along the small end with tissue forceps, completely tearing and spreading the amniotic layer flat, and tearing the vein and artery.

[0064] The tissue from which blood vessels had been removed was transferred to a 50 mL centrifuge tube containing 10 mL of digestive enzyme, and the tissue was cut with large surgical scissors into tissue chunks of 1 mm. 3 After sealing with a sealing membrane, the mixture was transferred to a shaking shaker at 37°C and digested with shaking at 60-80 revolutions / min for 2-3 hours.

[0065] The employed digestive enzymes were prepared according to the following formula: 20 mg / mL collagenase IV (Sigma-Aldrich), 40 mg / mL Dispase II (Sigma-Aldrich), 500 mM CaCL2 (Sigma-Aldrich), 20 mg / mL hyaluronic acid enzyme (Beijing Solabao Technology Co., Ltd.), 50 mM HEPES (buffer, BBI Life Science).

[0066] After the enzymatic digestion was completed, the surface of the test tube was disinfected with 75% ethanol, then the tube was transferred to a biological safety cabinet, and 30 mL of balanced salt solution HBSS was added to dilute and mix, followed by filtering through a 100 μm cell filtration screen.

[0067] The obtained filtrate was distributed into 15 mL centrifuge tubes, and about 10 mL of the filtrate was distributed per centrifuge tube, and centrifuged at 400 g at room temperature for 6 minutes. After the centrifugation was completed, the supernatant was slowly removed, and 1 mL of DMEM / F12 medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.) containing 10% fetal bovine blood substitute (prepared in Example 3) + 1% penicillin / streptomycin was added to each tube, and the precipitated cells were resuspended and combined into one tube, and then centrifuged at 400 g for 6 minutes. The supernatant was discarded, and 1 mL of DMEM / F12 medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.) containing 10% fetal bovine blood substitute (prepared in Example 3) + 1% penicillin / streptomycin was added and resuspended to obtain primary interstitial stem cells. The same volume of AO / PI reagent was added, mixed uniformly, and counted using a Countstar fluorescent counter. For details on the use of the automated cell counter, refer to the "Countstar Rigel S2 Automated Cell Counter Standard Operating Instructions" WWSC-0024-CC. The cell yield of primary umbilical cord stromal stem cells was statistically calculated.

[0068] The above experiment was repeated by replacing the fetal bovine serum substitute (prepared in Example 3) with fetal bovine serum (purchased from Excell Biotech (Taicang) Co., Ltd.), and the primary umbilical cord stromal stem cells were isolated and cultured, and the cell yield was statistically calculated. The results are shown in Figure 1.

[0069] Comparison of serum and serum substitutes in the culture of primary human hair follicle stromal stem cells Primary human hair follicle stromal stem cells were obtained by the method described in Example 2, and the primary human hair follicle stromal stem cells were isolated and cultured in DMEM / F12 medium containing 10% fetal bovine serum (purchased from Excell Biotech (Taicang) Co., Ltd.) and 10% fetal bovine serum substitute (prepared in Example 3), respectively, and the cell yield was statistically calculated. The results are shown in Figure 2.

[0070] Figures 1 and 2 show that the use of fetal bovine serum replacement in the culture medium can enhance the proliferation ability and increase the growth yield of primary cells, whether they are human umbilical cord stromal stem cells or human hair follicle stromal stem cells.

[0071] Example 5 Comparison of stem cell differentiation potential between serum and serum replacements The prepared fetal bovine serum substitute (prepared in Example 3) and commercial fetal bovine serum (purchased from Excell Biotech (Taicang) Co., Ltd.) were used in culturing to induce cell differentiation as follows:

[0072] Adipogenic differentiation and staining: First, hair follicle stromal stem cells were plated. Two wells of a 12-well plate were treated with gelatin, then dried at room temperature or in a cell incubator for 15 minutes. The hair follicle stromal stem cells harvested by passage were plated at a cell concentration of 2.5 × 10 5 The cells were placed in a 15 mL centrifuge tube and 10% fetal bovine serum substitute prepared in Example 3 or 10% commercial fetal bovine serum + human stromal stem cell serum-free medium (purchased from TBD Bioproducts Science and Technology Co., Ltd.) was added to a total volume of 2.5 mL, i.e., a final concentration of 1.0 × 10 5The cells were added to the wells of a coated 12-well plate at 1 mL of cell suspension per well, shaken 5 times, mixed evenly under a microscope, observed, and incubated in a 37°C incubator.

[0073] Next, adipogenic differentiation was induced. After the plated hair follicle stromal stem cells had grown to 90-100% confluence, the medium in the well was aspirated and discarded on a clean bench, and 1 mL of hair follicle stromal stem cell adipogenic differentiation induction medium (MesenCult Adipogenic Differentiation Medium (Human), purchased from STEMCELL Technologies) was added. Every 3 days, the old medium was discarded and replaced with new differentiation induction medium, and the medium was placed in a 37°C water bath and preheated for 15 minutes before use.

[0074] Finally, they were stained with Oil Red O. On the 14th day of inducing adipogenic differentiation, the medium in the well was aspirated and discarded, 1 mL of PBS was added, and the well was shaken three times back and forth, left and right, and the PBS was aspirated and discarded; 0.5 mL of 4% paraformaldehyde fixative was added to each well, and the well was shaken three times back and forth, left and right, to completely infiltrate the bottom of the well, and left to stand at room temperature for 15 minutes; The fixative was aspirated and discarded, 1 mL of PBS was added, and the well was shaken three times back and forth, left and right, to completely infiltrate the bottom of the well, and left to stand at room temperature for 8 minutes; 1 mL of isopropyl alcohol was added, then shaken, infiltrated, absorbed, and discarded; 1 mL of PBS was added, shaken three times, and discarded; 0.1 to 0.2 mL of PBS was added, and the staining situation was observed. The results of adipogenesis are shown in Figure 3, and the results of the adipogenesis rate (%) are shown in Table 2. [Table 2]

[0075] As is clear from Table 2 above, the use of a fetal bovine serum substitute in a culture medium can significantly enhance the adipogenic ability of hair follicle stromal stem cells, compared to commercial fetal bovine serum.

[0076] Differentiate chondrogenic cells and examine the expression of related genes: First, culture for induced differentiation was performed. Human hair follicle stromal stem cells were resuspended in 10% fetal bovine serum substitute prepared in Example 3 or 10% commercial fetal bovine serum + human stromal stem cell serum-free medium (purchased from TBD Bioproducts Science and Technology Co., Ltd.) to a cell concentration of 1 × 10 7 The cell concentration was adjusted to 10 cells / mL. In a clean bench, the above cell suspension was added to the inner surface of a 10 cm petri dish lid at a volume of 15 μL / drop, and 5 mL of PBS was added to the bottom of the petri dish to moisten it. The petri dish lid with the cell suspension dropped on it was carefully closed and incubated in a cell incubator for 24 hours, and the cell suspension on the petri dish lid gathered in a spherical shape. With a Pasteur pipette, the microspheres were carefully transferred to a low-adhesive twenty-four-well plate containing 1 mL chondrogenic differentiation induction medium (MesenCult ACF Chondrogenic Differentiation Basal Medium, purchased from STEMCELL Technologies). Every 3 days, 1-1.5 mL of new chondrogenic induction differentiation medium was replaced. On the third day, the cell spheres were collected in a 1.5 mL centrifuge tube. After washing twice with 1 mL of PBS and aspirating off the PBS, 50 μL of Trizol lysis solution (purchased from Nanjing Novozymes Biotechnology Co., Ltd.) was added to each tube, and the cell microspheres were ground using a high-speed tissue grinder for 15 seconds until the microspheres were finely crushed. Then, 950 μL of the lysis solution was added to each tube, and the tubes were repeatedly sprayed five times with a pipette tip, and vortexed for 30 seconds until the microspheres were completely broken up without any particles visible to the naked eye.

[0077] Next, we detected the expression of genes related to chondrogenic differentiation by extracting total RNA, reverse transcribing it to cDNA, and detecting gene expression by qPCR.

[0078] To the lysate of the cell microsphere sample, 100 μL of BCP (1-bromo 3-chloropropane) was added, mixed on a vortex for 15 seconds, placed on ice for 5 minutes, and centrifuged at 12000 rpm at 4°C for 15 minutes; the EP tube was carefully removed, and immediately approximately 500 μL of the upper aqueous phase was gently aspirated into a new 1.5 mL RNase-free EP tube, an equal volume of isopropyl alcohol was added, mixed by vortexing, and frozen at -80°C for 30 minutes or more; the tube was centrifuged at 12000 rpm at 4°C for 20 minutes, the supernatant was discarded, 1 mL of pre-cooled 75% ethanol was added, and the precipitate was washed by turning it upside down; the tube was centrifuged at 12000 rpm at 4°C for 5 minutes, the supernatant was discarded, the lid was opened, the precipitate was dried at room temperature, and 15-20 μL of DNase / RNase Free Add H2O, leave on ice for 5 minutes, vortex mix, centrifuge once to completely dissolve the precipitate; take 1μL of total RNA solution, measure RNA concentration, record the test results, and take 1μg of RNA for subsequent reverse transcription. The reverse transcription conditions were 25℃ for 10 minutes, 42℃ for 30 minutes, and 85℃ for 1 minute, and the above total RNA was reverse transcribed into cDNA. Primers and the corresponding qPCR system (PerfectStart Green qPCR SuperMix) were prepared to detect the expression status of genes involved in chondrogenic differentiation ("Research on Comprehensive Evaluation of In Vitro Chondrogenic Differentiation Ability of Human Mesenchymal Stem Cells", Zhang Kehua et al., China New Drug Journal, 2017,26(18),2196-2203). In this way, the percentage of up-regulated genes in the total genes involved in chondrogenic differentiation in human hair follicle stromal stem cells cultured in media containing fetal bovine serum substitutes or commercial fetal bovine serum was obtained, and the results are shown in Table 3 below. [Table 3]

[0079] Table 3 suggests that, compared with commercial fetal bovine serum, the use of fetal bovine serum substitutes in the culture medium enhances gene expression related to chondrogenic differentiation, thereby enhancing chondrogenic differentiation.

[0080] Although the present invention has been described with reference to specific examples, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the spirit and scope of the present invention. It is therefore intended by the appended claims to cover all such variations within the scope of the present invention.

Claims

1. A serum replacement comprising platelet lysate and plasma, the platelet lysate and plasma being derived from a bovine fetus or a newborn calf, preferably the serum replacement consisting of platelet lysate and plasma; preferably the bovine fetus is a bovine embryo between 5 and 8 months of age in a pregnant cow, and the newborn calf is a calf that is within 14 hours of birth and has not been fed.

2. 2. The serum substitute according to claim 1, which is obtained by dissolving platelets in platelet-rich plasma.

3. 3. The serum substitute according to claim 2, wherein the platelet-rich plasma is obtained by separating and removing blood cells other than platelets from fetal bovine whole blood or newborn bovine whole blood, Optionally, the serum replacement further comprises one or more of the following characteristics: The fetal or newborn bovine whole blood is anticoagulated; The separation is carried out using a centrifugation method, preferably a cryocentrifuge, which centrifugation is preferably a differential centrifugation; The platelet lysis is performed using the freeze-thaw method.

4. 2. A method for preparing a serum replacement according to claim 1, comprising lysis of platelets from fetal bovine or newborn bovine platelet plasma.

5. 5. The method of claim 4, comprising the steps of: a) isolating and removing blood cells other than platelets from fetal bovine whole blood or newborn bovine whole blood to obtain said platelet-rich plasma; preferably, said fetal bovine or newborn bovine whole blood is anticoagulated; b) Optionally, platelet lysis is followed by sterile grade filtration.

6. The method according to claim 5, wherein in step a), blood cells other than platelets are separated and removed by centrifugation, preferably by low-temperature centrifugation; preferably, the centrifugation speed is 400g to 2100g, e.g., 400g to 1000g, 1800g to 2100g, and the centrifugation time is 10 to 30 minutes, e.g., 10 to 25 minutes, 15 to 30 minutes; preferably, the low temperature is 10°C or lower, or 5°C or lower.

7. The centrifugation method is a differential centrifugation method, The differential centrifugation method includes at least one low-speed centrifugation step that can obtain a supernatant layer of platelet-rich plasma and a sediment layer enriched in blood cells other than platelets, and at least one high-speed centrifugation step that allows secondary separation of the plasma incompletely separated by the low-speed centrifugation from the blood cells; and mixing the supernatant collected prior to platelet lysis and / or incorporating the supernatant obtained by high speed centrifugation following platelet lysis; Preferably, the differential centrifugation includes at least one low speed centrifugation at a centrifugation speed of 400 g to 1000 g for a time period of 10 to 25 minutes; and The method of claim 6, comprising at least one high speed centrifugation at a centrifugation speed of 1800 g to 2100 g for a time period of 15 to 30 minutes.

8. 5. The method according to claim 4, wherein the platelet lysis is carried out using a freeze-thaw method, preferably the freeze-thaw method has one or more of the following characteristics: The number of freeze-thaw cycles is 3 to 7; Freezing time is at least 8 hours; The freezing temperature is below −20° C., preferably, the freezing temperature is −80° C.; The temperature of the melt is 0°C.

9. Use of the serum replacement according to any one of claims 1 to 3 in culturing stem cells, wherein the stem cells do not include cells isolated or obtained from a human embryo 14 days or more after fertilization, or cells developed in vivo; preferably, the stem cells are mesenchymal stem cells; more preferably, the stem cells are selected from human hair follicle mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells and human endometrial mesenchymal stem cells.

10. Use of the serum replacement according to any one of claims 1 to 3 in inducing stem cell differentiation, wherein the stem cells do not include cells isolated or obtained from a human embryo 14 days or more after fertilization, or cells developed in vivo; preferably, the stem cells are mesenchymal stem cells; more preferably, the stem cells are selected from human hair follicle mesenchymal stem cells, human umbilical cord mesenchymal stem cells, and human uterine mesenchymal stem cells; more preferably, the differentiation is, for example, adipogenic differentiation or chondrogenic differentiation.

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