Platelet lysate, its preparation method and application

Abstract

The application provides a platelet lysate and a preparation method and application thereof, and belongs to the technical field of cell culture. The application provides a platelet lysate, which comprises platelet lysate I and platelet lysate II; the platelet lysate I is derived from the blood of a newborn calf; the platelet lysate II is derived from the blood of an adult cow; and the volume ratio of the blood of the newborn calf to the blood of the adult cow is 1: (1.5-3). The platelet lysate has the characteristics that the blood of the newborn calf has a high growth factor content, and the factor spectrum of the blood of the adult cow is more mature and stable; the platelet lysate can simultaneously and efficiently promote the efficient proliferation of mesenchymal stem cells and maintain the stemness of the mesenchymal stem cells, and has important industrial value.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a platelet lysate, its preparation method, and its application. Background Technology

[0002] Fetal bovine serum (FBS) is the most widely used growth supplement in cell culture, but it has many inherent drawbacks, including large batch-to-batch variability, ethical controversies, the risk of contamination by potential animal-derived pathogens such as viruses and mycoplasma, and the potential to induce immune responses. These shortcomings severely limit the clinical application of FBS-based cells in regenerative medicine and cell therapy.

[0003] Platelet lysate (PL), as a potential alternative to fetal bovine serum (FBS), has become a hot topic in the field of cell culture due to its rich content of various growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF). However, in existing technologies, PL exhibits poor activity and cannot effectively promote the efficient proliferation of mesenchymal stem cells (MSCs) and maintain their stemness when used for MSC culture. Summary of the Invention

[0004] In view of this, the present invention provides a platelet lysate that can promote the efficient proliferation of mesenchymal stem cells and other adherent cells and maintain their stemness, and has important industrial value.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a platelet lysate, comprising platelet lysate I and platelet lysate II; The platelet lysate I is derived from the blood of newborn calves; The platelet lysate II is derived from the blood of an adult bovine. The volume ratio of the blood of the newborn calf to the blood of the adult calf is 1:(1.5~3).

[0006] Preferably, the volume ratio of the blood of the newborn calf to the blood of the adult calf is 1:2.

[0007] This invention provides a method for preparing the platelet lysate, comprising: obtaining platelet precipitate by differential centrifugation of blood; After washing and resuspending, the platelets were broken down by intermittent pulsed ultrasound to obtain platelet lysate. After removing cell membrane fragments and organelles from the platelet lysate, platelet lysate is obtained.

[0008] Preferably, the power of the intermittent pulsed ultrasonic fragmentation is 250~350 W, the time is 3~5 min, and the temperature is 2~8℃; The intermittent pulsed ultrasonic fragmentation consists of a working time of 1-3 seconds and an interval of 4-6 seconds.

[0009] Preferably, after removing cell membrane debris and organelles, the process further includes inactivation and filtration of pathogens; The method for inactivating pathogens includes irradiation treatment; the irradiation dose of the irradiation treatment is 25~40kGy.

[0010] The present invention provides a culture medium based on DMEM / F12, which also includes the platelet lysate or the platelet lysate prepared by the preparation method.

[0011] Preferably, the ratio of the basal culture medium to the platelet lysate is: 1-2 mL of platelet lysate extracted from blood is added to every 1 mL of basal culture medium.

[0012] This invention provides the application of the platelet lysate or the platelet lysate prepared by the preparation method or the culture medium in the culture of adherent cells.

[0013] Preferably, the adherent cells include mesenchymal stem cells; culturing mesenchymal stem cells includes promoting mesenchymal stem cell proliferation and / or maintaining mesenchymal stem cell stemness.

[0014] The present invention provides a method for culturing human umbilical cord-derived mesenchymal stem cells, comprising: culturing human umbilical cord-derived mesenchymal stem cells in the culture medium.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a platelet lysate, comprising platelet lysate I and platelet lysate II; platelet lysate I is derived from the blood of newborn calves; platelet lysate II is derived from the blood of adult cattle; the volume ratio of the newborn calves' blood to the adult cattle's blood is 1:(1.5~3). This invention, by mixing platelet lysates from newborn calves and adult cattle, combines the high growth factor content of newborn calves' blood with the more mature and stable factor profile of adult cattle's blood, resulting in a product with a more balanced effect on promoting proliferation and maintaining stemness. Examples of this invention demonstrate that culturing human umbilical cord-derived mesenchymal stem cells (MSCs) using the platelet lysate significantly improves the cell proliferation rate compared to the fetal bovine serum (FBS) group, significantly increases the number of cells proliferating at 120 hours, and significantly shortens the population doubling time. When cells were passaged to the P10 generation, the positivity rate of stem cell surface markers (CD73, CD90, and CD105) remained above 97.5%, while the positivity rate of stem cell surface markers in the FBS group decreased. At the same time, the senescence-related β-galactosidase activity of human umbilical cord-derived mesenchymal stem cells (MSCs) cultured with the platelet lysate was significantly reduced, the long-term expansion capacity was enhanced, and the osteogenic and adipogenic differentiation potential was also better maintained.

[0016] This invention provides a method for preparing platelet lysate, comprising: obtaining platelet precipitate by differential centrifugation of blood; washing and resuspending the platelet precipitate, followed by intermittent pulsed ultrasonic disruption to obtain platelet lysate; and removing cell membrane debris and organelles from the platelet lysate to obtain platelet lysate. This invention obtains high-purity, high-recovery platelets through low-temperature differential centrifugation, and the platelet lysate prepared by intermittent pulsed ultrasonic disruption can effectively promote the proliferation of mesenchymal stem cells and maintain their stemness. Examples of this invention demonstrate that the platelet lysate prepared by this method ensures nearly 100% disruption efficiency and over 95% retention of growth factor activity. Common freeze-thaw methods have low disruption efficiency and incomplete growth factor release (~70% release rate), while ultrasonic disruption, if parameters are not properly controlled, can easily lead to thermal denaturation and inactivation of growth factors, and it is difficult to completely remove cell debris and fibrinogen, the latter of which easily form fibrin clots during culture, interfering with cell growth. It is evident that the method for preparing platelet lysates is far superior to the freeze-thaw disruption method and the traditional ultrasonic continuous disruption. Attached Figure Description

[0017] Figure 1 This is a graph showing the results of a cell proliferation experiment. Detailed Implementation

[0018] This invention provides a platelet lysate, comprising platelet lysate I and platelet lysate II; The platelet lysate I is derived from the blood of newborn calves; The platelet lysate II is derived from the blood of an adult bovine. The volume ratio of the blood of the newborn calf to the blood of the adult calf is 1:(1.5~3).

[0019] In this invention, the volume ratio of the blood of the newborn calf to the blood of the adult calf is preferably 1:2. The breed of the newborn calf or the adult calf preferably includes Holstein cattle. The newborn calf preferably includes calf within 24 hours of birth. The age of the adult calf is preferably 2-5 years, more preferably 3-4 years. The adult calf preferably includes adult cows. The blood of the newborn calf has a high content of growth factors, while the blood of the adult calf has a more mature and stable factor profile. Embodiments of this invention demonstrate that by mixing platelet lysates from newborn calves and adult cattle and limiting their specific volume ratio, the proliferation-promoting factors (such as platelet-derived growth factor BB, PDGF-BB) and stemness-maintaining factors (such as insulin-like growth factor 1, IGF-1) in the obtained platelet lysates can achieve an optimal balance, effectively promoting the proliferation of mesenchymal stem cells and maintaining their stemness. The platelet lysate described in this invention contains platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and transforming growth factor-β (TGF-β), etc.

[0020] This invention provides a method for preparing the platelet lysate, comprising: obtaining platelet precipitate by differential centrifugation of blood; After washing and resuspending, the platelets were broken down by intermittent pulsed ultrasound to obtain platelet lysate. After removing cell membrane fragments and organelles from the platelet lysate, platelet lysate is obtained.

[0021] This invention obtains platelet precipitation by differential centrifugation of blood. Preferably, an anticoagulant is added to the collected blood. The anticoagulant preferably includes glucose citrate anticoagulant (ACD-A). The volume ratio of the anticoagulant to the blood is preferably 1:7-11, more preferably 1:8-10, and most preferably 1:9. This ratio helps maintain the integrity and activity of platelets while achieving limited anticoagulation, avoiding platelet damage or osmotic pressure changes caused by excessive anticoagulant, thereby ensuring a high recovery rate and activity of platelets obtained from subsequent centrifugation. In this invention, the platelet lysate includes a mixture of platelet lysate I and platelet lysate II. The mixing can be performed at various times: first, mixing newborn calf blood and adult calf blood during the blood collection phase; second, mixing platelet precipitates from newborn calf blood and adult calf blood after differential centrifugation; third, mixing platelet lysates from newborn calf blood and adult calf blood after intermittent pulsed sonication; fourth, mixing platelet lysates from newborn calf blood (with cell membrane debris and organelles removed) and platelet lysates from adult calf blood (with cell membrane debris and organelles removed) after removing cell membrane debris and organelles; and fifth, mixing platelet lysate I and platelet lysate II after removing cell membrane debris and organelles. Preferably, the present invention involves mixing the collected newborn calf blood and adult calf blood before differential centrifugation. Mixing the blood from both sources before differential centrifugation ensures that platelets from both sources undergo identical separation conditions, avoiding operational errors introduced by separate processing. This achieves uniform mixing from the source, ensuring a strictly consistent ratio of the two platelet types in each subsequent processing unit. This simplifies the process, reduces operational steps, and lowers the risk of contamination. The two plasma components interact before separation, potentially producing a better synergistic effect. The differential centrifugation preferably involves a first centrifugation of the blood to obtain an upper plasma layer, a middle leukocyte layer, and a lower erythrocyte layer; followed by a second centrifugation of the upper plasma layer to obtain a second supernatant and a second precipitate, the second precipitate being platelet precipitate. The centrifugal force for the first centrifugation is preferably 180-220 g, more preferably 190-210 g, and even more preferably 200 g. The first centrifugation time is preferably 10-30 min, more preferably 15-25 min, and even more preferably 20 min. The centrifugal force for the second centrifugation is preferably 700-900 g, more preferably 750-950 g, and even more preferably 800 g. The second centrifugation time is preferably 20-40 min, more preferably 25-35 min, and even more preferably 30 min. The differential centrifugation temperature is preferably 3.5-4.5℃, and even more preferably 4℃.The low-temperature differential centrifugation parameters described in this invention are beneficial for obtaining platelets with high purity and high recovery rate, while protecting the effective components in the platelets. The platelet lysate prepared can effectively promote the proliferation of mesenchymal stem cells and maintain the stemness of mesenchymal stem cells.

[0022] After obtaining the platelet precipitate, the platelet precipitate is washed and resuspended. The washing reagent preferably includes phosphate-buffered saline (PBS solution). Washing removes residual plasma proteins. After washing, the platelet precipitate is resuspended, and the resuspending reagent preferably includes PBS solution. The amount of resuspending reagent used is measured by the volume of blood used to obtain the platelet precipitate; the volume ratio of the resuspending reagent to the blood is preferably 1:(25~35), more preferably 1:(28~32), and even more preferably 1:30. The washing or resuspending temperature is preferably 3.5~4.5℃, more preferably 4℃. The washing and resuspending parameters of this invention, combined with the subsequent platelet lysis step, allow the prepared platelet lysate to retain more effective components, effectively promoting the proliferation of mesenchymal stem cells and maintaining their stemness.

[0023] After resuspension, platelet lysate is obtained by intermittent pulsed ultrasound disruption. The power of the intermittent pulsed ultrasound disruption is preferably 250-350 W, more preferably 280-320 W, and even more preferably 300 W. The duration of the intermittent pulsed ultrasound disruption is preferably 3-5 min, more preferably 3.5-4.5 min, and even more preferably 4 min. The temperature of the intermittent pulsed ultrasound disruption is preferably 2-8℃. The intermittent pulsed ultrasound disruption is preferably performed for 1-3 seconds with an interval of 4-6 seconds, more preferably 2 seconds with an interval of 5 seconds or 3 seconds with an interval of 5 seconds. The parameters of the intermittent pulsed ultrasound disruption of this invention ensure complete disruption of the platelet membrane and full release of growth factors, while minimizing thermal denaturation, which is beneficial for the prepared platelet lysate to promote mesenchymal stem cell proliferation and maintain their stemness. This invention compares the effects of different disruption methods (traditional repeated freeze-thaw method, continuous ultrasonic disruption, 200W low-temperature pulsed ultrasonic disruption, 400W low-temperature pulsed ultrasonic disruption, and 300W low-temperature pulsed ultrasonic disruption) on platelet disruption efficiency and growth factor retention rate. The results show that 300W low-temperature pulsed ultrasonic disruption has the best effect on platelet disruption efficiency and growth factor retention rate.

[0024] After removing cell membrane debris and organelles from the platelet lysate, platelet lysate is obtained. The method for removing cell membrane debris and organelles preferably includes high-speed centrifugation. The centrifugal force of the high-speed centrifugation is preferably 11000-13000 g, more preferably 12000 g. The high-speed centrifugation time is preferably 40-50 min, more preferably 45 min. The high-speed centrifugation temperature is preferably 3.5-4.5℃, more preferably 4℃. The high-speed centrifugation can thoroughly precipitate cell membrane debris and organelles. After removing cell membrane debris and organelles, platelet lysate with removed cell membrane debris and organelles is obtained. Preferably, the process further includes inactivating and filtering pathogens in the platelet lysate after removing cell membrane debris and organelles. The method for inactivating pathogens preferably includes irradiation treatment. Pre-filtration of the platelet lysate with removed cell membrane debris and organelles is preferred before irradiation treatment. The pore size of the pre-filter is preferably 0.45 μm, which can remove residual cell membrane debris, protein aggregates, and particles larger than 0.45 μm. The filtrate obtained after pre-filtration is subjected to irradiation treatment. The preferred radiation source for irradiation treatment is cobalt-60 irradiation, and the preferred irradiation dose is... The irradiation treatment can range from 25 to 40 kGy, and can be 30 to 35 kGy, with 25 kGy being the most preferred. The irradiation treatment can remove any viruses and mycoplasma that may be present. After the irradiation treatment, filtration is preferably performed. The pore size of the filter membrane used for filtration is preferably 0.22 μm. The filtration can sterilize the product. Platelet lysate is obtained after sterilization. This method of pathogen inactivation and filtration, without using chemical reagents or affecting the activity of growth factors, greatly improves the biosafety of the product and meets the regulatory requirements for the preparation of clinical-grade cell products.

[0025] In this embodiment of the invention, the contents of the key growth factors PDGF-BB, vascular endothelial growth factor (VEGF), and IGF-1 in the platelet lysate prepared by the method are 195±7 ng / mL, 142±6.5 pg / mL, and 91.7±3.5 ng / mL, respectively. The platelet lysate prepared by the method of this invention exhibits high activity, stable composition, safety, and harmlessness. It can simultaneously promote the efficient proliferation of MSCs and maintain their stemness, thus possessing significant industrial value.

[0026] The present invention provides a culture medium based on DMEM / F12, which also includes the platelet lysate or the platelet lysate prepared by the preparation method.

[0027] In this invention, the culture medium preferably further includes serum. The serum content is preferably ≤5%. The preferred ratio of the basal culture medium to the platelet lysate is: 1-3 mL of platelet lysate extracted from blood per 1 mL of basal culture medium, more preferably 1.2-2.5 mL of platelet lysate extracted from blood per 1 mL of basal culture medium, even more preferably 1.4-1.6 mL of platelet lysate extracted from blood per 1 mL of basal culture medium, and most preferably about 1.5 mL of platelet lysate extracted from blood per 1 mL of basal culture medium. The preferred volume ratio of the basal culture medium to the platelet lysate is 1:9-30, more preferably 1:15-25, and even more preferably 1:19. In this embodiment of the invention, the platelet lysate prepared by the method accounts for 5%-10% of the volume of the basal culture medium. This invention compared the culture effects of 5% and 10% additions, and the results showed that both effectively promoted MSC proliferation and maintained their stemness, with the 5% addition showing slightly better results. Therefore, the most preferred addition amount in this invention is approximately 5% (corresponding to the addition of platelet lysate extracted from approximately 1.5 mL of blood to every 1 mL of basal culture medium). Examples of this invention demonstrate that the culture medium with the aforementioned addition ratio can effectively promote the proliferation of mesenchymal stem cells and maintain their stemness.

[0028] This invention provides the application of the platelet lysate or the platelet lysate prepared by the preparation method or the culture medium in the culture of adherent cells.

[0029] The platelet lysate described in this invention contains platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and transforming growth factor-β (TGF-β). These growth factors, by binding to corresponding receptors on the cell membrane (such as tyrosine kinase receptors and serine / threonine kinase receptors), activate downstream signaling pathways (such as the PI3K / Akt and MAPK / ERK pathways), and can universally promote the adhesion, survival, proliferation, and migration of various adherent cells.

[0030] In this invention, the adherent cells include mesenchymal stem cells. The mesenchymal stem cells preferably include human umbilical cord-derived mesenchymal stem cells and / or adipose-derived mesenchymal stem cells. The mesenchymal stem cells preferably include P3 generation or higher mesenchymal stem cells, more preferably P3-P15 generation mesenchymal stem cells, and most preferably P5-P10 generation mesenchymal stem cells. The culture of mesenchymal stem cells preferably includes promoting mesenchymal stem cell proliferation and / or maintaining mesenchymal stem cell stemness. Embodiments of this invention demonstrate that the culture medium can effectively promote the proliferation of P3-P10 generation mesenchymal stem cells and maintain their stemness; at the P10 generation, the positive rate of stem cell surface markers remains at 99%.

[0031] The present invention provides a method for culturing human umbilical cord-derived mesenchymal stem cells, comprising: culturing human umbilical cord-derived mesenchymal stem cells in the culture medium.

[0032] In this invention, the mesenchymal stem cells are preferably the same as those used in the above applications, and will not be described again here. Embodiments of this invention demonstrate that the method can effectively promote the proliferation of mesenchymal stem cells and maintain their stemness.

[0033] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: Preparation method of platelet lysate The preparation method of platelet lysate involves the following steps: S1. Aseptic Blood Collection and Mixing: Under aseptic conditions, 100 mL of blood from newborn Holstein calves within 2 hours of birth and 200 mL of blood from healthy adult Holstein cows aged 2 years were collected via the bovine jugular vein. The collected blood was transferred separately to blood collection bags containing citrate glucose anticoagulant (ACD-A) at a volume ratio of 1:9. The anticoagulant calf blood and anticoagulant adult bovine blood were then mixed thoroughly in a sterile connector at a volume ratio of 1:2, yielding 333.3 mL of mixed anticoagulant bovine blood (300 mL of total blood and approximately 33.3 mL of total ACD-A).

[0035] S2. Low-temperature differential centrifugation for platelet separation: The mixed anticoagulated bovine blood was dispensed into 50mL centrifuge tubes and centrifuged at 200×g for 20min in a high-speed centrifuge at 4℃. After centrifugation, the upper platelet-rich plasma (PRP) layer was carefully aspirated with a sterile pipette, avoiding contact with the middle white blood cell layer. Combine the collected PRP to obtain approximately 120 mL of PRP. Transfer the PRP to a new centrifuge tube. Centrifuge the collected PRP at 4°C with a centrifugal force of 800 × g for 30 min. After centrifugation, slowly and thoroughly discard the supernatant to obtain a pale yellow platelet precipitate.

[0036] S3. Platelet pellet washing and resuspension: Gently resuspend the platelet pellet in 40 mL of pre-cooled, sterile phosphate-buffered saline (PBS) at 4°C, and wash by centrifugation at 800 × g for 30 min at 4°C to remove residual plasma proteins. After centrifugation, discard the supernatant, and finally resuspend the platelet pellet in 10 mL of PBS until concentrated. Transfer the concentrated platelet suspension to a 15 mL conical tube to obtain a concentrated platelet suspension.

[0037] S4. Low-Temperature Pulsed Ultrasonic Disruption: The concentrated platelet suspension was placed in an ice-water bath and disrupted using a Branson SFX550 ultrasonic cell disruptor equipped with a 3mm probe. Specific parameters were: power 300 W, pulse mode with 2 seconds of operation followed by 5 seconds of interval, total ultrasonic time 4 minutes, and the ice bath was maintained throughout the process to ensure the sample temperature remained between 2 and 8°C. After disruption, platelet lysate was obtained.

[0038] S5. Debris removal and clarification: Immediately centrifuge the platelet lysate at 12000×g for 45 min at 4°C to completely precipitate cell membrane debris and organelles, and collect the supernatant.

[0039] S6. Pathogen inactivation and filtration: The supernatant was pre-filtered through a 0.45 μm filter membrane, and then the filtrate was sterilized using cobalt-60 with an irradiation dose of [missing value]. 25 kGy was applied to remove any potential viruses and mycoplasma. Finally, the platelet lysate was filtered through a 0.22 μm sterile membrane for terminal sterilization. The filtrate was then aliquoted into 2 mL sterile cryovials to obtain platelet lysate, which was stored at -80°C.

[0040] S7. Quality Control: Platelet lysate samples were tested, including sterility testing, mycoplasma testing, and endotoxin testing (requirement <1 EU / mL). The concentrations of key growth factors (PDGF-BB, VEGF, and IGF-1, purchased from Cusabio, catalog numbers CSB-EL017709BO, CSB-EL016078BO, and CSB-EL011128BO, respectively) were quantitatively determined using ELISA kits as the standard for batch release. The quality control results for platelet lysates are shown in Table 1.

[0041] Mycoplasma was detected by PCR. Specifically, 1 mL of cell culture supernatant was collected, centrifuged at 13000 g for 5 min, the precipitate was resuspended, and then heated to 90℃ for 15 min for lysis. Specific primers were designed based on the conserved region of mycoplasma 16S rRNA, and mycoplasma DNA was detected by PCR amplification.

[0042] Primer A: 5'-GGCGAATGGGTGAGTAACACG-3' (SEQ ID NO: 1); Primer B: 5'-CGGATAACGCTTGCGACCTATG-3' (SEQ ID NO: 2); PCR reaction conditions: Pre-denaturation: 94℃ for 3 min; 30 cycles: 94℃ 25 s → 58℃ 45 s → 72℃ 45 s; Final extension: 72℃ for 7 minutes.

[0043] Table 1. Quality control results of platelet lysates

[0044] Example 2 Optimization of platelet fragmentation methods To screen for the optimal platelet disruption method, the effects of different disruption methods (traditional repeated freeze-thaw cycle, continuous ultrasonic disruption, 200W low-temperature pulsed ultrasonic disruption, 400W low-temperature pulsed ultrasonic disruption, and 300W low-temperature pulsed ultrasonic disruption) on platelet disruption efficiency and growth factor retention rate were compared. Based on the different disruption methods, the following 5 groups were established.

[0045] Group 1: Platelet lysates were prepared according to the preparation method of mixed-source platelet lysates in Example 1, with the only difference being that S4 was the traditional repeated freeze-thaw method: the concentrated platelet suspension was frozen at -80°C for 12 hours, and then rapidly thawed in a water bath at 37°C. The freeze-thaw process was repeated 3 times to obtain platelet lysates. Group 2: Platelet lysates were prepared according to the method described in Example 1, with the only difference being that S4 involved continuous ultrasonic disruption: the concentrated platelet suspension was disrupted using a Branson SFX550 ultrasonic cell disruptor equipped with a 3mm probe. Specific parameters were: power 300W, continuous operation for 4 minutes, and no ice-water bath during the entire process. After disruption, platelet lysate was obtained. Group 3: Platelet lysates were prepared according to the preparation method of mixed-source platelet lysates in Example 1, the only difference being that the ultrasonic power in S4 was 200W; Group 4: Platelet lysates were prepared according to the preparation method of mixed-source platelet lysates in Example 1, the only difference being that the ultrasound power in S4 was 400W; Group 5: Platelet lysates were prepared using the method described in Example 1 for preparing mixed-source platelet lysates.

[0046] After obtaining platelet lysates, the platelet fragmentation efficiency and growth factor retention rate of each group were measured. Using a fully automated hematology analyzer (Sysmex XN-1000), the number of intact platelets in the concentrated platelet suspension before ultrasonic fragmentation (denoted as N_before) and the number of residual intact platelets in the platelet lysate after ultrasonic fragmentation and before high-speed centrifugation (denoted as N_after) were determined. The fragmentation efficiency was calculated as follows: Fragmentation efficiency (%) = [(N_before - N_after) / N_before] × 100%. The growth factor retention rate was determined using an ELISA kit (bovine-specific kit, Cusabio) to detect the concentrations of PDGF-BB, VEGF, and IGF-1 in each group of samples. The growth factor retention rate was calculated as follows: Retention rate (%) = [Growth factor concentration in the test group / Theoretical total concentration] × 100%. The results are shown in Table 2.

[0047] Table 2 Platelet fragmentation efficiency and growth factor retention rate in each group

[0048] The results showed that Group 1, using the traditional repeated freeze-thaw method, had a platelet fragmentation efficiency of only 72%; Group 2, using continuous ultrasonic fragmentation, resulted in growth factor denaturation due to lack of temperature control, leading to a large amount of fibrin clots, with a platelet fragmentation efficiency of 90%; Group 3, using 200W pulsed ultrasonic fragmentation, had a fragmentation efficiency of only 85%; Group 4, using 400W pulsed ultrasonic fragmentation, resulted in excessive platelet membrane fragmentation due to excessive power, which was difficult to remove by subsequent centrifugation and interfered with MSC cell adhesion; Group 5, using controllable ultrasonic fragmentation, achieved a platelet fragmentation efficiency of 99.5%, with no protein clots, a fragmentation rate of 99%, and the least amount of fragments, without affecting cell adhesion. Therefore, the specific ultrasonic fragmentation parameters (ice bath, short pulse) in Group 5 (Example 1) are the core for high-purity, high-recovery platelets, ensuring nearly 100% fragmentation efficiency and 96% retention of growth factor activity, far superior to the freeze-thaw method.

[0049] Comparative Example 1 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, except that in S1, 100 mL of blood from a Holstein calf less than 2 hours old and 200 mL of blood from a healthy adult Holstein cow aged 2 years were replaced with 300 mL of blood from a Holstein calf less than 2 hours old.

[0050] Comparative Example 2 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, except that in S1, 100 mL of blood from a Holstein calf less than 2 hours old and 200 mL of blood from a healthy adult Holstein cow aged 2 years were replaced with 300 mL of blood from a healthy adult Holstein cow aged 2 years.

[0051] Comparative Example 3 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, except that in S1, 100 mL of blood from a Holstein calf less than 2 hours old and 200 mL of blood from a healthy adult Holstein cow aged 2 years were replaced with 150 mL of blood from a Holstein calf less than 2 hours old and 150 mL of blood from a healthy adult Holstein cow aged 2 years.

[0052] Comparative Example 4 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, except that in S1, 100 mL of blood from a Holstein calf less than 2 hours old and 200 mL of blood from a healthy adult Holstein cow aged 2 years were replaced with 60 mL of blood from a Holstein calf less than 2 hours old and 240 mL of blood from a healthy adult Holstein cow aged 2 years old.

[0053] Comparative Example 5 A high-quality fetal bovine serum (FBS) (purchased from Gibco).

[0054] Comparative Example 6 A commercially available human platelet lysate, PLTMax (purchased from Sigma-Aldrich), catalog number: SCM141.

[0055] Example 3 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, with the only difference being the use of different batches of Holstein bovine blood (batch 2) to prepare different batches of PL.

[0056] Example 4 Platelet lysates were prepared according to the method for preparing mixed-source platelet lysates in Example 1, with the only difference being the use of different batches of Holstein bovine blood (batch 3) to prepare different batches of PL.

[0057] Example 5: Application of platelet lysate in cultured human umbilical cord-derived MSCs Culture method for human umbilical cord-derived MSCs: Fresh human umbilical cords from healthy mothers after cesarean section are collected and repeatedly washed with PBS containing 1% penicillin and antibiotics to remove blood. The umbilical cord is then cut into 1-2 mm pieces. 3The tissue blocks were digested with 0.1% type I collagenase at 37°C for 3 hours. After digestion was terminated, the cells were filtered through a 70μm cell sieve, and the cell suspension was collected. The suspension was then diluted with 1×10⁻⁶ cells per cell culture medium. 4 pcs / cm 2 Human umbilical cord MSCs were seeded at a density in T75 culture flasks, and DMEM / F12 medium containing 10% fetal bovine serum was added. The flasks were then incubated at 37°C in a 5% CO2 incubator. After 7-10 days of culture, when the cell confluence reached 80%, the cells were digested with 0.25% trypsin and passaged to obtain generation P0 human umbilical cord MSCs. Generation P0 cells were continuously passaged to generation 3 (generation P3), and generation P3 human umbilical cord MSCs were collected for subsequent experiments.

[0058] P3 generation human umbilical cord MSCs were seeded at the same density in different 96-well plates and cultured. The different 96-well plates were prepared with DMEM / F12 medium supplemented with different components. Based on the different components, they were divided into: Example 1 group, Example 3 group, Example 4 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group (FBS group), and Comparative Example 6 group (human PL group). The volume percentage of each component in the DMEM / F12 medium was 5%–10%, as detailed in Table 3. PLTMax added heparin to the medium to prevent coagulation during culture preparation.

[0059] The samples were cultured continuously for 5 days at 37℃, 5% CO2, and saturated humidity. A set of wells was sampled daily, and the absorbance (OD) was measured using CCK-8 reagent. 450nm To detect cell proliferation, cells were cultured continuously for 5 days at 37℃, 5% CO2, and saturated humidity. A set of wells was sampled daily, and the absorbance (OD) was measured using CCK-8 reagent. 450 Cell proliferation was assessed using nmol / 2.5 nm (nm). At passage P4, a portion of cells from each group were used for osteogenic induction. P4 cells were cultured at 2 × 10⁻⁶ cells / mL. 4 pcs / cm 2Cells were seeded at a density in 6-well plates. When cell confluence reached 70%, the medium was replaced with osteogenic induction medium (DMEM / F12 basal medium containing 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate sodium, and 50 μM ascorbate phosphate). The medium was changed every 3 days for 21 consecutive days of induction. After induction, cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and then stained with 0.2% alizarin red staining solution (pH 4.2) at room temperature for 30 min. After washing with distilled water, the formation of calcium nodules was observed under a microscope, and differentiation potential was assessed by alizarin red staining. When the remaining cells were cultured to passages P5 and P10, they were digested with trypsin and then labeled with CD73, CD90, and CD105 (all purchased from BD Biosciences, catalog numbers: CD73-PE 550257, CD90-PE555596, CD105-PE 562408) by flow cytometry. Cell stem cell markers were detected, and the positive rates of stem cell surface markers (CD73, CD90, and CD105) were calculated. The positive rate was calculated using the formula: Positive rate (%) = (CD73 + CD90 + CD105 + number of cells / total number of cells) × 100%. The results of cell proliferation experiments, stem cell marker detection, and differentiation potential assays for each group are shown in Table 3.

[0060] Table 3. Components added to DMEM / F12 culture medium and detection results in each group.

[0061] Note: This indicates that P < 0.05. P < 0.001, and ns indicates no significant difference.

[0062] Cell proliferation experiments showed that the platelet lysate group prepared from mixed blood in Example 1 had the highest cell proliferation efficiency, significantly shortened population doubling time, and a significantly higher cell proliferation rate than the fetal bovine serum group and the commercially available human platelet lysate group. The cell proliferation rate of the platelet lysate group prepared from calf blood in Comparative Example 1 was significantly lower than that in Example 1. The cell proliferation rate of the platelet lysate group prepared from adult cow blood in Comparative Example 2 was significantly lower than that in Example 1. The cell proliferation rate of the platelet lysate group prepared from mixed blood (calf blood to adult cow blood volume ratio of 1:1) in Comparative Example 3 was significantly lower than that in Comparative Example 4 (calf blood to adult cow blood volume ratio of 1:4), and the cell proliferation efficiency of Comparative Example 4 was 20% lower than that of Example 1.

[0063] Flow cytometry analysis of stem cell markers showed that at passage P5, the positive rates of all three markers in the platelet lysate group prepared from mixed blood in Example 1 were >99%, while those in the fetal bovine serum group and the commercially available human platelet lysate group decreased to approximately 95% and 97%, respectively. In Comparative Example 1, the stem cell marker positive rate was only 90% after passage to passage P5; in Comparative Example 2, the positive rate was below 90% after passage to passage P5. In Example 1, the positive rate of stem cell surface markers remained at 99% after continuous passage to passage P10. In Comparative Example 3, the positive rate was 89% after passage to passage P10; in Comparative Example 4, the positive rate was 92%, lower than in Example 1. In Example 1, the positive rate remained at 99% after passage to passage P10.

[0064] Alizarin red staining results showed that the platelet lysate group prepared by mixing blood in Example 1 formed the most calcium nodules and stained the most deeply, indicating that its osteogenic differentiation potential was best maintained.

[0065] In summary, the results demonstrate that the platelet lysate prepared by the method of this invention can effectively promote the proliferation of mesenchymal stem cells (MSCs), maintain their stemness, and exhibit excellent batch-to-batch and passage stability. Specifically, when MSCs were cultured using the platelet lysate of this invention and passaged continuously to the P10 generation, the cell proliferation rate and the positive rate of surface markers did not significantly decrease with increasing passage number. The coefficients of variation (CV) of key quality indicators (growth factor content) and cell culture effects (cell proliferation rate, positive rate) of the platelet lysate prepared using different batches (from different individual bovine sources) of blood according to the method of this invention were less than 15%, indicating robust process and consistent product quality.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A platelet lysate, characterized in that, Including platelet lysate I and platelet lysate II; The platelet lysate I is derived from the blood of newborn calves; The platelet lysate II is derived from the blood of an adult bovine. The volume ratio of the blood of the newborn calf to the blood of the adult calf is 1:(1.5~3).

2. The platelet lysate according to claim 1, characterized in that, The volume ratio of the blood of the newborn calf to the blood of the adult calf is 1:

2.

3. A method for preparing the platelet lysate according to claim 1, characterized in that, include: Platelet precipitate was obtained by differential centrifugation of blood. After washing and resuspending, the platelets were broken down by intermittent pulsed ultrasound to obtain platelet lysate. After removing cell membrane fragments and organelles from the platelet lysate, platelet lysate is obtained.

4. The preparation method according to claim 3, characterized in that, The power of the intermittent pulsed ultrasonic fragmentation is 250~350 W, the time is 3~5 min, and the temperature is 2~8℃; The intermittent pulsed ultrasonic fragmentation consists of a working time of 1-3 seconds and an interval of 4-6 seconds.

5. The preparation method according to claim 3 or 4, characterized in that, The process of removing cell membrane debris and organelles also includes inactivation and filtration of pathogens; The method for inactivating pathogens includes irradiation treatment; the irradiation dose of the irradiation treatment is 25~40kGy.

6. A culture medium, characterized in that, The medium is based on DMEM / F12 and also includes the platelet lysate as described in claim 1 or 2 or the platelet lysate prepared by any one of the preparation methods described in claims 3 to 5.

7. The culture medium according to claim 6, characterized in that, The ratio of the basal culture medium to the platelet lysate is: 1-2 mL of platelet lysate extracted from blood is added to every 1 mL of basal culture medium.

8. The use of the platelet lysate of claim 1 or 2, or the platelet lysate prepared by any one of claims 3 to 5, or the culture medium of claim 6 or 7 in the culture of adherent cells.

9. The application according to claim 8, characterized in that, The adherent cells include mesenchymal stem cells; culturing mesenchymal stem cells includes promoting mesenchymal stem cell proliferation and / or maintaining mesenchymal stem cell stemness.

10. A method for culturing human umbilical cord-derived mesenchymal stem cells, characterized in that, include: Human umbilical cord-derived mesenchymal stem cells were cultured using the culture medium described in claim 6.

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