Cell drug and preparation method thereof

Abstract

The invention relates to the technical field of cell preparation, and particularly discloses a cell drug and a preparation method thereof.The cell drug comprises mesenchymal cells and pharmaceutically acceptable auxiliary materials; the mesenchymal cell is characterized in that the expression of SOX2, DPPA4 and MYCN genes is negative; the auxiliary material comprises a sodium chloride solution containing albumin; the concentration of the sodium chloride solution is 0.1%-2%; the content of the albumin in the sodium chloride solution is 0.1%-10%. The cell medicine is simple in composition and good in auxiliary material compatibility, the motility of active ingredients can be protected, and the safety and reliability of the cell medicine are remarkably improved by optimizing the preparation process.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell preparation and relates to a cell medicine and a preparation method thereof. Background Art

[0002] Mesenchymal cells, first isolated from bone marrow by Friedenstein in 1982, are a specialized cell population found within mesenchymal tissues and possess the ability to divide and differentiate. Research has shown that mesenchymal cells are widely distributed in various tissues, including bone marrow, umbilical cord, and adipose tissue, and demonstrate significant potential in tissue regeneration and repair.

[0003] Although mesenchymal cells have demonstrated good safety in multiple clinical trials, their therapeutic efficacy remains uncertain. Studies have shown that in animal experiments, intravenous infusion of mesenchymal cells may accumulate in the pulmonary microvascular bed, leading to pulmonary circulatory disorders and even serious adverse reactions such as respiratory failure.

[0004] Therefore, in order to improve the clinical availability of mesenchymal cells, it is urgent to optimize their preparation process and improve the composition of cell products to ensure their safety, efficacy and consistency. Summary of the Invention

[0005] In response to the deficiencies of the prior art and actual needs, the present invention provides a cell medicine and a preparation method thereof to obtain a mesenchymal cell medicine with high safety.

[0006] In a first aspect, the present invention provides a cell medicine comprising mesenchymal cells and a pharmaceutically acceptable excipient; The mesenchymal cells are SOX2 、 DPPA4 and MYCN gene expression negative; The excipients include sodium chloride solution containing albumin; The concentration of the sodium chloride solution is 0.1% to 2%; The content of the albumin in the sodium chloride solution is 0.1% to 10%.

[0007] The mesenchymal cells are CLDN11 Gene expression was positive.

[0008] The present invention identifies the active ingredients of cell medicine and discovers the self-renewal and differentiation markers unique to stem cells SOX2 、 DPPA4 and MYCN The expression of stromal cells was negative. CLDN11 Gene expression was positive and the active ingredients met the criteria for stromal cell identification.

[0009] Preferably, the concentration of the sodium chloride solution is 0.1% to 2%, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%. Preferably, 0.9% sodium chloride injection is used to dilute the cells to maintain the internal and external osmotic pressure balance of the cells.

[0010] Preferably, the content of albumin in the sodium chloride solution is 0.1% to 10%, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8. %, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9% or 10%. The present invention uses a sodium chloride solution containing albumin as an adjuvant for cell medicine, which plays a very important role in maintaining the biological activity and structural integrity of mesenchymal cells.

[0011] The present invention comprehensively considers factors such as in vivo efficacy, toxicological experiments, and clinically intended administration routes and dosages, and selects an injection as the dosage form of the cell drug.

[0012] Preferably, the mesenchymal cells are derived from one or more of fat, umbilical cord, placenta, and bone marrow.

[0013] Preferably, the number of mesenchymal cells in the cell medicine is 10 5 ~10 9 / mL, for example 1×10 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL, 5×10 5 / mL, 6×10 5 / mL, 7×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 / mL, 5×10 6 / mL, 6×10 6 / mL, 7×10 6 / mL, 8×10 6 / mL, 9×10 6 / mL, 1×10 7 / mL, 2×10 7 / mL, 3×10 7 / mL, 4×10 7 / mL, 5×10 7 / mL, 6×10 7 / mL, 7×10 7 / mL, 8×10 7 / mL, 9×10 7 / mL, 1×10 8 / mL, 2×10 8 / mL, 3×10 8 / mL, 4×10 8 / mL, 5×10 8 / mL, 6×10 8 / mL, 7×10 8 / mL, 8×10 8 / mL, 9×10 8 / mL, 1×10 9 / mL, 2×10 9 / mL, 3×10 9 / mL, 4×10 9 / mL, 5×10 9 / mL, 6×10 9 / mL, 7×10 9 / mL, 8×10 9 / mL or 9×10 9 pieces / mL.

[0014] Preferably, the content of DMSO in the cell medicine is less than 0.5%.

[0015] In a second aspect, the present invention provides a method for preparing the cell medicine according to the first aspect, comprising the steps of resuscitating and washing a mesenchymal cell stock solution to obtain mesenchymal cells, which are the active ingredient of the cell medicine: After the mesenchymal cell stock solution is revived, it is washed with a washing solution and resuspended with an auxiliary material to obtain the cell medicine.

[0016] Preferably, the washing solution comprises sodium chloride solution, and optionally albumin.

[0017] Preferably, the concentration of the sodium chloride solution is 0.1% to 2%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2%, preferably 0.9% sodium chloride injection.

[0018] The content of albumin in the sodium chloride solution is 0.1% to 10%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%. , 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9% or 10%.

[0019] Preferably, the number of washings is 1 to 10 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times.

[0020] Preferably, the volume ratio of the washing solution to the mesenchymal cell stock solution is (0.1~1):1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1.

[0021] As a preferred embodiment, the method for preparing the cell medicine comprises: After the mesenchymal cell stock solution is revived, it is washed 1 to 10 times with a 0.1% to 2% sodium chloride solution, the volume ratio of the washing solution to the mesenchymal cell stock solution is (0.1 to 1):1, and resuspended with a sodium chloride solution containing 0.1% to 10% albumin to obtain the cell drug.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The cell medicine of the present invention has a simple composition and good compatibility of excipients, can protect the activity of the active ingredients of mesenchymal cells, and ensures that the cell activity of the active ingredients of the cell medicine is as high as 90% or more within a storage time of 6 hours by optimizing the preparation process, thereby significantly improving the safety and reliability of the cell medicine. DETAILED DESCRIPTION

[0023] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0024] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0025] Example 1 Adipose tissue and umbilical cord tissue were obtained from donors, from which mesenchymal cells were extracted.

[0026] Adipose-derived mesenchymal cells (AD-MSCs) were prepared as follows: the collected adipose tissue was washed several times with physiological saline, and the floating adipose tissue layer was digested with 0.3 U / mL collagenase at 37°C for 1 hour. After digestion, the adipose tissue was centrifuged and filtered, and 2 mL was inoculated into a T75 culture flask and cultured with αMEM (Gibco) supplemented with 5% UltraGRO Advanced (HELIOS) for 5-10 days (P0 cells). When the cell confluence reached 50-70%, the cells were digested with 1×TrypLE (Gibco) and passaged into new culture flasks (from P0 to P1). The same culture medium was then used to culture the cells at 5000 cells / cm 2The density of the cells is 5000 cells / cm2, and the cells of the 2nd generation (P2) and the 5th generation (P5) are used to establish the seed cell bank and the working cell bank, respectively. The cells are recovered from the working cell bank, suspended in the physiological saline containing 1% albumin, and prepared into the final product with the concentration of 1x10 6 The mesenchymal cells are obtained by freezing the final product.

[0027] The umbilical cord mesenchymal cells (UC-MSCs) are prepared as follows: the umbilical cord tissue is washed with PBS solution, cut into small pieces, uniformly spread in a T75 culture bottle, incubated in a CO2 incubator for 10 minutes, and cultured overnight using the culture medium of aMEM (Gibco) supplemented with 5% UltraGRO Advanced (HELIOS). The culture medium is replaced every 3 days (P0 cells). When the cells around the tissue pieces are covered, the cells are digested using 1x TrypLE (Gibco), and the cells are subcultured into new culture bottles (from P0 to P1). The subsequent culture is performed using the same culture medium with the density of 5000 cells / cm 2 The cells of the 2nd generation (P2) and the 5th generation (P5) are used to establish the seed cell bank and the working cell bank, respectively. The cells are recovered from the working cell bank, suspended in the physiological saline containing 1% albumin, and prepared into the final product with the concentration of 1x10 6 The mesenchymal cells are obtained by freezing the final product.

[0028] Example 2 After the mesenchymal cell stock solution prepared in Example 1 is recovered, it is washed once with 2% sodium chloride solution, the volume ratio of the washing solution to the cell mesenchymal cell stock solution is 1:1, and it is resuspended in sodium chloride solution containing 0.1% albumin to obtain the cell drug.

[0029] Example 3 After the mesenchymal cell stock solution prepared in Example 1 is recovered, it is washed twice with 1.5% sodium chloride solution, the volume ratio of the washing solution to the cell mesenchymal cell stock solution is 0.8:1, and it is resuspended in sodium chloride solution containing 0.5% albumin to obtain the cell drug.

[0030] Example 4 After the mesenchymal cell stock solution prepared in Example 1 is recovered, it is washed three times with 1.2% sodium chloride solution, the volume ratio of the washing solution to the cell mesenchymal cell stock solution is 0.7:1, and it is resuspended in sodium chloride solution containing 1% albumin to obtain the cell drug.

[0031] Example 5 After the mesenchymal cell stock solution prepared in Example 1 was revived, it was washed five times with a 0.9% sodium chloride solution, the volume ratio of the washing solution to the mesenchymal cell stock solution was 0.5:1, and resuspended with a sodium chloride solution containing 2% albumin to obtain the cell drug.

[0032] Example 6 After the mesenchymal cell stock solution prepared in Example 1 was revived, it was washed 7 times with a 0.5% sodium chloride solution, the volume ratio of the washing solution to the mesenchymal cell stock solution was 0.3:1, and resuspended with a sodium chloride solution containing 3% albumin to obtain the cell drug.

[0033] Example 7 After the mesenchymal cell stock solution prepared in Example 1 was revived, it was washed 8 times with a 0.2% sodium chloride solution, the volume ratio of the washing solution to the mesenchymal cell stock solution was 0.2:1, and resuspended with a sodium chloride solution containing 8% albumin to obtain the cell drug.

[0034] Example 8 The mesenchymal cell stock solution prepared in Example 1 was revived and washed 10 times with a 0.1% sodium chloride solution, the volume ratio of the washing solution to the mesenchymal cell stock solution being 0.1:1, and resuspended with a sodium chloride solution containing 10% albumin to obtain the cell drug.

[0035] The cell viability of the cellular drugs prepared in Examples 2-8 was measured at 0 h and 6 h. The cell suspension was diluted with 0.9% sodium chloride injection and thoroughly mixed with 0.4% trypan blue dye at a volume ratio of 9:1. 10 μL of the mixture was pipetted into the counting chamber of a disposable counting plate and observed under a 10x objective lens. The total number of live and dead cells in each of the four squares was recorded. Cell viability was calculated according to the following formula.

[0036] Viable cell rate (%) = total number of viable cells / (total number of viable cells + total number of dead cells) × 100%.

[0037] The results are shown in Table 1. The cell viability of the cell medicines in each example was ≥80%. After storage at 2-8°C for 6 hours, the cell viability of the cell medicines in each example was ≥80%. Among them, the cell viability of the cell medicine in Example 5 was slightly higher than that of the cell medicines in other examples, indicating that the optimized preparation process in Example 5 helps improve the safety and reliability of the cell medicine.

[0038] Table 1 The present application is illustrated by the above examples, but the present application is not limited to the above detailed methods, i.e. it is not meant that the present application must rely on the above detailed methods to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A cell medicine, characterized in that: comprising mesenchymal cells and pharmaceutically acceptable excipients; The mesenchymal cells are SOX2 、 DPPA4 and MYCN gene expression negative; The excipients include sodium chloride solution containing albumin; The concentration of the sodium chloride solution is 0.1% to 2%; The content of the albumin in the sodium chloride solution is 0.1% to 10%.

2. The cell medicine according to claim 1, characterized in that The mesenchymal cells are CLDN11 Gene expression was positive.

3. The cell medicine according to claim 1, characterized in that The mesenchymal cells are derived from one or more of fat, umbilical cord, placenta, and bone marrow.

4. The cell medicine according to claim 1, characterized in that The number of mesenchymal cells in the cell medicine is 10 5 ~10 9 pieces / mL.

5. The cell medicine according to claim 1, characterized in that The content of DMSO in the cell medicine is less than 0.5%.

6. The method for preparing the cell medicine according to any one of claims 1 to 5, characterized in that: include: After the mesenchymal cell stock solution is revived, it is washed with a washing solution and resuspended with an auxiliary material to obtain the cell medicine.

7. The preparation method according to claim 6, characterized in that The washing solution comprises sodium chloride solution and, optionally, albumin.

8. The preparation method according to claim 7, characterized in that The concentration of the sodium chloride solution is 0.1% to 2%; The content of the albumin in the sodium chloride solution is 0.1% to 10%.

9. The preparation method according to claim 6, characterized in that The number of washings is 1 to 10 times.

10. The preparation method according to claim 6, characterized in that The volume ratio of the washing solution to the mesenchymal cell stock solution is (0.1~1):1.

Images