A method for separating and freezing giant panda colostrum cells and cryoprotective solution

Abstract

The present invention discloses a method for separating and freezing panda colostrum cells and a cryoprotectant, which belongs to the field of giant panda colostrum freezing preservation technology. The method comprises the following components at a final concentration: 1.5-3 wt% honey, 1.5-3 wt% sucrose, 20-30 wt% giant panda milk, 0.5-1.5×10 6 Cell equivalents / mL of giant panda colostrum cell lysate, and normal saline. The present invention also developed an active preparation specifically for giant panda colostrum for use in conjunction with frozen colostrum. This preparation uses the natural cell components in giant panda colostrum as the core raw material, and is supplemented with the above-mentioned cryoprotectant. After freezing, the preparation can be stably stored in a liquid nitrogen environment. When used, it is thawed and then added back to the frozen colostrum, which can effectively replenish the cell components lost in the frozen colostrum during storage, making it closer to the biological activity of fresh colostrum, thereby improving the feeding effect of frozen colostrum.

Description

Technical Field

[0001] The invention belongs to the technical field of giant panda colostrum cryopreservation, and particularly relates to a giant panda colostrum cell separation and cryopreservation method and a cryoprotectant solution. Background Art

[0002] Ensuring timely and adequate colostrum intake is crucial for giant panda cub survival. With the continuous advancement of captive panda breeding technology, the frequency of twin births has increased. Female giant pandas cannot produce enough milk to feed two cubs simultaneously, necessitating the use of frozen colostrum to ensure the survival of newborn cubs. However, this technique has a drawback: the cells in the frozen colostrum die before feeding.

[0003] Fresh colostrum contains a large number of functional living cells. These cells not only promote immune system development and strengthen the pup's immunity, but also, while the intestinal barrier of newborn pups is still incomplete, some cells can cross the intestines and enter the circulatory system. They then migrate to various organs and develop into cells with corresponding functions, providing comprehensive support for the pup's growth. Given the relatively early developmental stage of newborn giant panda cubs, their immune systems are extremely underdeveloped and highly susceptible to pathogenic infections. Therefore, it is hypothesized that the cellular components in giant panda colostrum are even more crucial to the cub's early development than those of other animals. However, during giant panda rearing, colostrum is primarily preserved by freezing in liquid nitrogen. While liquid nitrogen freezing effectively preserves colostrum's nutrients, it damages the cells, killing approximately 95% of the living cells. Furthermore, frozen colostrum must be pasteurized before use, further eliminating any remaining living cells.

[0004] The core difference between frozen colostrum and fresh colostrum lies in whether it contains live cells, and this difference is likely the key reason why frozen colostrum is less effective than fresh colostrum in feeding. Fresh colostrum can provide cubs with more comprehensive and effective immune support during the critical period after birth, significantly reducing the risk of illness. Analysis of feeding records and health status of giant panda cubs revealed that within the first seven days of birth, giant panda cubs fed frozen colostrum experienced significantly more illnesses than cubs fed only fresh colostrum. This result suggests that in the early stages after birth, fresh colostrum has a more significant effect on promoting the development of the cub's immune system than frozen colostrum. Therefore, how to effectively preserve the live cells in fresh colostrum and successfully add them to frozen colostrum becomes the key to enhancing the efficacy of frozen colostrum and compensating for its shortcomings.

[0005] At room temperature, fresh colostrum cells have a very short shelf life (usually no more than one day). Even in a refrigerated environment (0-4°C), their shelf life is extremely limited (generally only two to three days), and they are highly susceptible to bacterial contamination during this process. In contrast, ultra-low temperature freezing technology enables long-term, stable cell preservation (perhaps for years or more). Given the temporary and uncertain nature of giant panda rearing, the cells need to be readily available and flexible, ready for immediate use when needed. Therefore, freezing giant panda colostrum cells in liquid nitrogen to create an active colostrum cell preparation is undoubtedly the most ideal method for its current application, satisfying both the long-term preservation needs of the cells and their ready availability during the rearing process.

[0006] However, traditional cell freezing solutions usually contain chemical components such as DMSO, which are not suitable for direct feeding. Therefore, they must be thawed and cleaned in the laboratory before use. This process not only increases the complexity of the operation, but also leads to a delay in application, making it difficult to meet the urgent needs of immediate feeding of giant pandas. The feeding of giant pandas is mainly the responsibility of professional keepers, who need a more convenient and efficient operation method, and the complicated cell recovery procedure will obviously restrict their daily work. In addition, for those breeding institutions that lack laboratory sterile conditions, this operation method is almost impossible to implement, which greatly limits its widespread application in actual production. Summary of the Invention

[0007] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for separating and freezing giant panda colostrum cells and a cryoprotectant. The present invention also develops an active preparation specifically for giant panda colostrum for use in conjunction with frozen colostrum. The preparation uses the natural cell components in giant panda colostrum as the core raw material, and is supplemented with a cryoprotectant that is safe, edible, and suitable for direct ingestion by giant pandas. After freezing, the preparation can be stably stored in a liquid nitrogen environment. When used, it is thawed and then added back to the frozen colostrum, which can effectively supplement the cell components lost in the frozen colostrum during storage, making it closer to the biological activity of fresh colostrum, thereby improving the feeding effect of frozen colostrum.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0009] The object of the present invention is to provide a giant panda colostrum cell cryoprotective solution comprising the following components at final concentrations:

[0010] 1.5~3wt% honey, 1.5~3wt% sucrose, 20~30wt% giant panda milk, 0.5~1.5×10 6 Cell equivalents / mL of giant panda colostrum cell lysate and normal saline.

[0011] Furthermore, the following components are included at final concentrations:

[0012] 2.5~3wt% honey, 2.5~3wt% sucrose, 25~30wt% giant panda milk, 1~1.5×10 6 Cell equivalents / mL of giant panda colostrum cell lysate and normal saline.

[0013] Furthermore, the giant panda's normal milk is the milk secreted from the 7th to 30th day.

[0014] Furthermore, giant panda colostrum cell lysate is prepared from P1 to P3 generations of giant panda colostrum cells.

[0015] Furthermore, the preparation process of giant panda colostrum cell lysate is as follows:

[0016] Giant panda colostrum cells of generations P1 to P3 were collected, resuspended in sterile water, and ultrasonically lysed. The cells were then centrifuged at 10,000 to 12,000 g for 10 to 15 minutes, and the supernatant was collected to obtain giant panda colostrum cell lysate.

[0017] Furthermore, ultrasound was performed using 80% power, with a fragmentation time of 5 s and a pause time of 5 s for 12 cycles.

[0018] Another object of the present invention is to provide a method for separating frozen colostrum cells from giant panda colostrum, comprising the following steps:

[0019] (1) Using a giant panda colostrum cell separation solution to centrifuge and separate colostrum cells from giant panda colostrum; the giant panda colostrum cell separation solution includes the following components at final concentrations:

[0020] 2.5–5 mM N-acetylcysteine, 0.5–1 wt% BSA, 200–240 UI / mL penicillin, 200–250 μg / mL streptomycin, 0.5–1.2 μg / mL amphotericin, and PBS;

[0021] (2) The isolated colostrum cells were cultured in a modified DMEM low-glucose medium; the modified DMEM low-glucose medium included: 10% KSR (KnockOut serum replacement), 0.5-1×10 6 Cell equivalent / mL giant panda umbilical cord mesenchymal stem cell lysate, 85-100 UI / mL penicillin, 75-100 μg / mL streptomycin;

[0022] (3) The colostrum cells cultured in step (2) are placed in the above-mentioned giant panda colostrum cell cryoprotective solution and then frozen for storage.

[0023] Furthermore, the giant panda colostrum cell separation solution in step (1) includes the following components at final concentrations:

[0024] 5 mM N-acetylcysteine, 1 wt% BSA, 200 UI / mL penicillin, 200 μg / mL streptomycin, 0.5 μg / mL amphotericin, and PBS.

[0025] Furthermore, the modified DMEM low-glucose medium in step (2) includes: 10% KSR, 1×10 6 Cell equivalent / mL giant panda umbilical cord mesenchymal stem cell lysate, 100 UI / mL penicillin, 100 μg / mL streptomycin.

[0026] Furthermore, the preparation method of giant panda umbilical cord mesenchymal stem cell lysate is as follows:

[0027] Giant panda umbilical cord mesenchymal stem cells were resuspended in sterile water, ultrasonically lysed, and centrifuged at 10,000-12,000 g for 10-15 min. The supernatant was collected to obtain the giant panda umbilical cord mesenchymal stem cell lysate.

[0028] Another object of the present invention is to provide a use of the giant panda colostrum cell cryoprotective solution in the preparation of a giant panda colostrum active preparation.

[0029] Beneficial effects of the present invention:

[0030] The present invention has developed a cryopreservation solution containing only sucrose, honey, and giant panda cell extracts. This solution can be consumed with the cells, meeting the cryopreservation requirements of colostrum cells while ensuring the safety of giant pandas, greatly simplifying the use process. Ultimately, through in-depth research and systematic summary of the dynamic changes in cells in normal colostrum from a large number of giant pandas, combined with the age characteristics of the pups, a precise and appropriate dosage of colostrum cells was determined, thus scientifically and effectively solving the problem of cellular component loss in frozen colostrum.

[0031] The present invention also developed an active formulation specifically for giant panda colostrum, for use with frozen colostrum. This formulation uses the natural cellular components of giant panda colostrum as its core ingredient, supplemented with a safe, edible cryoprotectant suitable for direct ingestion by giant pandas. After freezing, the formulation can be stably stored in a liquid nitrogen environment. Upon use, the formulation is thawed and then added to the frozen colostrum, effectively replenishing the cellular components lost during storage, bringing the colostrum's biological activity closer to that of fresh colostrum, thereby enhancing the feeding effect of frozen colostrum.

[0032] There are reports on freezing and adding colostrum cells to giant pandas (separating the cells from the colostrum and preserving them separately, then adding them to frozen milk when feeding). This technique has two major drawbacks: First, the centrifugation process separates some of the colostrum's proteins, which are then lost during cell washing. Consequently, the colostrum cannot be reused for panda rearing and must be discarded, a significant waste of the precious colostrum. Second, this technique only preserves colostrum, not amplifies it. This waste of resources makes it difficult to use with existing frozen colostrum banks and, therefore, difficult to apply to panda rearing. In contrast, the present invention supplements the missing living cell components, making it more similar in composition to fresh colostrum, thereby improving its effectiveness in rearing pandas. Furthermore, the colostrum cell isolation and production process has a resource-amplifying effect: colostrum cells obtained from 1 mL of fresh colostrum can be cultured and used to add to approximately 4.5 L of frozen milk, fully meeting daily panda rearing needs. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0034] Example 1 Study on the Isolation Effect of Giant Panda Colostrum Cells

[0035] Collect 2 mL of giant panda colostrum (1-7 days after birth) through acclimation milking. Take 1 mL each and add 29 mL of conventional separation buffer and giant panda colostrum cell separation buffer. Place the sample on ice and quickly return to the laboratory (within 2 hours). Pre-cool the centrifuge to 4°C, centrifuge at 600g for 20 minutes, and discard the supernatant. Wash once with 10 mL of each of the two corresponding separation buffers, centrifuge at 600g for 10 minutes, and discard the supernatant. The components of the two separation buffers are as follows:

[0036] Conventional separation solution: PBS, 200 UI / mL penicillin, 200 μg / mL streptomycin, and 0.5 μg / mL amphotericin.

[0037] Giant panda colostrum cell isolation medium: PBS, 5 mM N-acetylcysteine, 1% BSA, 200 UI / mL penicillin, 200 μg / mL streptomycin, and 0.5 μg / mL amphotericin.

[0038] After separation, the cells were uniformly resuspended in 1 mL of DMEM (low glucose) medium, and the collected cells were stained with trypan blue and counted using a counting plate to detect cell viability and number. The results are shown in Table 1.

[0039] Table 1 Cell viability

[0040]

[0041] Note: n=9, *There was a significant difference between the conventional separation fluid group and the giant panda colostrum cell separation fluid group, Student's t-test, *P<0.05 .

[0042] The results in Table 1 show that the giant panda colostrum cell separation solution provided by the present invention can significantly improve the viability of the obtained cells without affecting the number of separated cells.

[0043] Example 2 Establishment of a culture system for giant panda colostrum cells

[0044] The cells were uniformly isolated using Giant Panda Colostrum Cell Separation Medium (Example 1, Giant Panda Colostrum Cell Separation Medium). The pellets were resuspended in four different culture media and seeded into 48-well plates for culture. Because the cultured cells were intended for feeding giant panda cubs, fetal bovine serum (FBS) was replaced with KSR (Knockout Serum Replacement) to ensure feeding safety. The effects of the four culture media on giant panda colostrum cell culture were compared.

[0045] Method 1: DMEM (low glucose), 10% KSR (KnockOut Serum Replacement), 100 UI / mL penicillin, 100 μg / mL streptomycin.

[0046] Method 2: DMEM (low glucose), 10% KSR (KnockOut Serum Replacement), giant panda umbilical cord mesenchymal stem cell lysate (1×10 4 cell equivalent / mL), 100 UI / mL penicillin, and 100 μg / mL streptomycin.

[0047] Method 3: DMEM (low glucose), 10% KSR (KnockOut Serum Replacement), giant panda umbilical cord mesenchymal stem cell lysate (1×10 5 cell equivalent / mL), 100 UI / mL penicillin, and 100 μg / mL streptomycin.

[0048] Method 4: DMEM (low glucose), 10% KSR (KnockOut Serum Replacement), giant panda umbilical cord mesenchymal stem cell lysate (1×10 6cell equivalent / mL), 100 UI / mL penicillin, and 100 μg / mL streptomycin.

[0049] The preparation method of giant panda umbilical cord mesenchymal stem cell lysate is as follows:

[0050] 5×10 7 Giant panda umbilical cord mesenchymal stem cells were resuspended in 0.5 mL of pre-cooled sterile water and lysed by ultrasonication (80% power, 5 s disruption, 5 s pause; 12 cycles). After ultrasonication, the sample was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was collected and stored in aliquots, each designated as 1 × 10 8 Cell equivalent / mL.

[0051] The initial inoculated cell number was 1×10 4 After 24 hours, cells adhered to the culture medium and the corresponding culture medium was replaced daily. Cells were counted after 5 days of culture. Since the initial cell number was the same, the count results after 5 days can reflect the cell proliferation status. The cell proliferation factor after 5 days = the number of cells after 5 days of culture / the initial number of cells inoculated (i.e., 1×10 4 cells), and the results are shown in Table 2.

[0052] Table 2 Proliferation status

[0053]

[0054] Note: The differences between the two groups are significant. One-Way ANOVA with Tukey's Multiple Comparison Test. *P <0.05.

[0055] The results in Table 2 show that the present invention found that after adding giant panda umbilical cord mesenchymal stem cell lysate, the proliferation effect of giant panda colostrum cells was greatly improved. 5 The effect is best, continue to increase the concentration to 1×10 6 The effect of the increase is not significant.

[0056] Example 3 Study on the cell generation range for preparing giant panda colostrum cell preparation

[0057] Cells have limitations in culture passages in vitro, primarily manifesting as aging and decreased activity, which gradually reduce their applicability. To further confirm the optimal passage for giant panda colostrum cells, we conducted continuous cell culture experiments to investigate cell proliferation at different passages and determine the appropriate passage range for preparing colostrum cell preparations.

[0058] The cell proliferation of 3 colostrum cells at passages P2-P9 was measured. The specific steps are as follows:

[0059] Take P2 cells and calculate 2×10 4 Cells were seeded in 24-well plates and passaged after 4 days of culture. 4 Cells / well were inoculated into 24-well plates for culture (denoted as P3). The culture medium used was giant panda colostrum cell culture medium (Example 2, Method 3). 4-day proliferation factor = number of cells after 4 days of culture / initial number of cells inoculated (i.e. 2×10 4 The results are shown in Table 3.

[0060] Table 3 4-day proliferation times

[0061]

[0062] Note: The differences between the two groups are significant. One-Way ANOVA with Tukey's Multiple Comparison Test. *P <0.05.

[0063] According to the test results in Table 3, from the perspective of cell proliferation, in the giant panda colostrum cell culture system (Example 2, Method 3), the proliferation of giant panda colostrum cells from P2 to P5 is relatively stable, while from P8 to P9, the cell proliferation ability drops sharply.

[0064] In actual production, panda colostrum cells are primarily expanded and frozen for storage at passages P1 to P2. After thawing the stored panda colostrum cells, they can be expanded in large quantities at passages P3 to P5 for the preparation of colostrum cell preparations. Generally speaking, 1 mL of panda colostrum cells can produce approximately 6 P2 cells (each containing approximately 5×10 5 After P2, based on the cumulative 4-day proliferation of cells in each generation, the number of cells in 1 mL of colostrum can increase by about 4,500 times when expanded to P5, which is sufficient to meet the cell addition requirements of 4.5 L of frozen colostrum.

[0065] Example 4 Development and Effect Evaluation of Edible-Grade Cryopreservation Solution for Giant Pandas

[0066] This study compared the cryoprotective effects of three safe, edible freezing solutions on giant panda colostrum cells (10% DMSO + 90% FBS as a control). Honey and sucrose are both common foods, while giant panda milk and giant panda colostrum cell lysate are both derived from giant panda milk.

[0067] Method 1: 10% DMSO, 90% FBS.

[0068] Method 2: Normal saline, 3% honey, 3% sucrose.

[0069] Method 3: Normal saline, 3% honey, 3% sucrose, 30% giant panda milk (lactation within 7 days to 1 month).

[0070] Method 4: Normal saline, 3% honey, 3% sucrose, 30% giant panda normal milk (lactation within 7 days to 1 month), giant panda colostrum cell lysate (P1-P3, 1×10 6 cell equivalents / mL).

[0071] The preparation method of giant panda colostrum cell lysate is as follows:

[0072] Take 5×10 6 Giant panda colostrum cells (P1-P3) were resuspended in 0.5 mL of pre-cooled sterile water and lysed by ultrasonication (80% power, 5 s disruption, 5 s pause; 12 cycles). After ultrasonication, the sample was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was collected and recorded as 1 × 10 7 Cell equivalent / mL.

[0073] When cells were frozen, 1×10 6 cells, and resuspended in 0.1 mL freezing buffer (cells / freezing buffer = 1 × 10 7 The cell suspension was then placed in a programmed freezing box and frozen overnight in a -80°C freezer. The next day, the frozen cells were removed from the -80°C freezer and transferred to liquid nitrogen for long-term storage. The cells were thawed six months after freezing. During thawing, the frozen cells were quickly removed from the liquid nitrogen and immediately placed in a 37°C water bath with rapid shaking to ensure rapid thawing. The thawed cells were then added to 5 mL of Colostrum Cell Culture Medium, centrifuged at 600 g for 10 minutes, and the supernatant discarded. The cells were resuspended in 1 mL of Colostrum Cell Culture Medium and stained with trypan blue to determine cell viability. The results are shown in Table 4.

[0074] Table 4 Cell viability after freezing

[0075]

[0076] Note: The differences between the two groups are significant. One-Way ANOVA with Tukey's Multiple Comparison Test. *P <0.05.

[0077] The results in Table 4 show that the giant panda colostrum cell cryopreservation solution (Method 4) provided by the present invention can significantly improve the freezing effect of giant panda colostrum cells, with the average cell viability reaching 55% after freezing. Although there is still a certain gap compared with the 97% cell viability of the control group, the formula used in the present invention does not contain any toxic or harmful substances or non-giant panda-derived protein components, and can be used directly for feeding after thawing without additional processing.

[0078] Example 5 Determining the amount of giant panda colostrum cells added

[0079] The giant panda colostrum cell active additive developed by the present invention is specially designed for situations where frozen milk is needed for giant panda cubs within one month after birth due to insufficient fresh milk supply. This additive is intended to significantly enhance the feeding efficacy of frozen milk, provide better support for cubs in the early stages of development, and thus optimize the rearing effect. Previous studies have found that the number of cells in giant panda milk varies significantly between different lactation days. Therefore, in order to further optimize the cell addition strategy, it is necessary to deeply explore the dynamic changes in the number of cells in giant panda milk, so as to achieve scientific and reasonable cell addition.

[0080] Collect 1 mL of giant panda milk by milking and add 29 mL of giant panda colostrum cell separation solution (Example 1, Giant Panda Colostrum Cell Separation Solution). Return the sample to the laboratory (within 2 hours) and centrifuge at 600g for 20 minutes. Discard the supernatant, wash each well once with 10 mL of separation solution, and centrifuge at 600g for 10 minutes. After separation, resuspend the cells in DMEM (low-glucose) medium and count the cells. The results are shown in Table 5.

[0081] Table 5 Cell content

[0082]

[0083] Note: The differences between the two groups are significant. One-Way ANOVA with Tukey's Multiple Comparison Test. *P <0.05.

[0084] Nine healthy giant pandas were selected for cell count monitoring at different lactation time points. The results in Table 5 show that during the first 30 days of lactation, the cell count of giant pandas showed regular changes. The colostrum cell count was approximately 110,000 cells / mL from D1 to D3, and approximately 60,000 cells / mL from D4 to D7. The cell content showed a significant downward trend on D7, and then gradually stabilized at 20,000 cells / mL.

[0085] Example 6 Preparation and Use of Giant Panda Colostrum Cell Preparation

[0086] Giant panda colostrum cells were isolated using a giant panda colostrum cell separation solution (Example 1, Giant Panda Colostrum Cell Separation Solution) according to the method described in Example 1. Giant panda colostrum cells were expanded in vitro using a giant panda colostrum cell culture system (Example 2, Method 3). Giant panda colostrum cells from passages P2-P5 (the passage range is determined in Example 3) were resuspended in a giant panda colostrum cell cryopreservation solution (Example 4, Method 4) at a specific ratio. The specific addition amount was calculated based on the cell content of giant pandas at different lactation stages (Example 5) and the cryopreservation efficiency (Example 4, 55%). Preparations were produced according to three steps. The results are shown in Table 6.

[0087] Table 6 Formulation design

[0088]

[0089] 5×10 giant panda colostrum cells of generation P3-P6 were cultured 6 resuspended in 0.5 mL giant panda colostrum cell freezing solution (Example 4, Method 4) to prepare 1×10 7 The cell concentration of the preparation is 100 cells / mL (the cell concentration of the finished preparation).

[0090] 1. Giant panda colostrum cell active preparation: Add 20 μL of preparation stock solution to a 1 mL cryopreservation tube. Suitable for giant panda cubs 1-3 days old.

[0091] Stage 2 Giant Panda Colostrum Cell Active Preparation: Add 11 μL of preparation stock solution to a 1 mL cryovial. Suitable for giant panda cubs 4-7 days old.

[0092] Stage 3 Giant Panda Colostrum Cell Active Preparation: Add 4 μL of the preparation stock solution to a 1 mL cryovial. Suitable for giant panda cubs aged 8-30 days.

[0093] The outside of the cryovial is labeled with information about the giant panda colostrum cell preparation, including the cell segment number, cell source, cell passage number, production batch, and production date. Before use, each batch of cell preparation undergoes mycoplasma testing (conventional PCR testing), bacterial testing (conventional bacterial culture testing), and safety verification for feeding to newborn mice (three individuals, each fed for three days).

[0094] When using, take one tube of the corresponding preparation according to the age of the pups, quickly place it in a 37°C water bath, incubate for 10 seconds, then add 1 mL of rewarmed (37°C) frozen milk (which can be pasteurized in advance) to the freezing tube containing the colostrum cell preparation, mix well and then use it to feed the pups.

[0095] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A giant panda colostrum cell cryoprotective solution, characterized in that: Include the following components at final concentrations: 1.5~3wt% honey, 1.5~3wt% sucrose, 20~30wt% giant panda milk, 0.5~1.5×10 6 cell equivalents / mL of giant panda colostrum cell lysate and normal saline; The giant panda colostrum cell lysate is prepared from P1 to P3 generation giant panda colostrum cells, and the preparation process is as follows: Giant panda colostrum cells of generations P1 to P3 were collected, resuspended in sterile water, and ultrasonically lysed. The cells were then centrifuged at 10,000 to 12,000 g for 10 to 15 minutes, and the supernatant was collected to obtain giant panda colostrum cell lysate.

2. The giant panda colostrum cell cryoprotective solution according to claim 1, characterized in that: Include the following components at final concentrations: 2.5~3wt% honey, 2.5~3wt% sucrose, 25~30wt% giant panda milk, 1~1.5×10 6 Cell equivalents / mL of giant panda colostrum cell lysate and normal saline.

3. The giant panda colostrum cell cryoprotective solution according to claim 1 or 2, characterized in that: Giant panda milk is the milk secreted from the 7th to 30th day.

4. The giant panda colostrum cell cryoprotective solution according to claim 1, characterized in that Ultrasonication was performed at 80% power, with a 5-s on-off and a 5-s pause for 12 cycles.

5. A method for separating frozen colostrum cells from giant panda colostrum, characterized in that: The following steps are involved: (1) Using a giant panda colostrum cell separation solution to centrifuge and separate colostrum cells from giant panda colostrum; the giant panda colostrum cell separation solution includes the following components at final concentrations: 2.5–5 mM N-acetylcysteine, 0.5–1 wt% BSA, 200–240 UI / mL penicillin, 200–250 μg / mL streptomycin, 0.5–1.2 μg / mL amphotericin, and PBS; (2) Cultivating the isolated colostrum cells in a modified DMEM low-glucose medium; The modified DMEM low-glucose culture medium includes: 10% KSR, 0.5-1×10 6 Cell equivalent / mL giant panda umbilical cord mesenchymal stem cell lysate, 85-100 UI / mL penicillin, 75-100 μg / mL streptomycin; (3) The colostrum cells cultured in step (2) are placed in the giant panda colostrum cell cryoprotective solution according to any one of claims 1 to 4, and then cryopreserved.

6. The method according to claim 5, characterized in that The giant panda colostrum cell separation solution in step (1) includes the following components at final concentrations: 5 mM N-acetylcysteine, 1 wt% BSA, 200 UI / mL penicillin, 200 μg / mL streptomycin, 0.5 μg / mL amphotericin, and PBS.

7. The method according to claim 5, characterized in that The modified DMEM low-glucose medium in step (2) includes: 10% KSR, 1×10 6 Cell equivalent / mL giant panda umbilical cord mesenchymal stem cell lysate, 100 UI / mL penicillin, 100 μg / mL streptomycin.

8. The method according to claim 5 or 7, characterized in that The preparation method of giant panda umbilical cord mesenchymal stem cell lysate is as follows: Giant panda umbilical cord mesenchymal stem cells were resuspended in sterile water, ultrasonically lysed, and placed in a centrifuge. Centrifugation was performed at 10,000-12,000 g for 10-15 min, and the supernatant was collected to obtain the giant panda umbilical cord mesenchymal stem cell lysate.

9. Use of the giant panda colostrum cell cryoprotective solution according to any one of claims 1 to 4 in the preparation of a giant panda colostrum active preparation.

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