Mitochondria preservation composition and preparation method thereof

Abstract

The invention belongs to the technical field of mitochondrial preservation, and particularly relates to a mitochondrial preservation composition and a preparation method thereof.The mitochondrial preservation composition obtained through clinical common liquid including human serum albumin, glucose and normal saline according to a controlled proportion can maintain the mitochondrial structure and functional activity and is suitable for in-vivo transplantation and preservation, and the mitochondrial preservation effect is good. Immunogenicity and toxicity are remarkably reduced, and clinical safety standards are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of mitochondrial preservation, and in particular relates to a mitochondrial preservation composition and a preparation method thereof. Background Art

[0002] Mitochondrial preservation solutions are designed to maintain mitochondrial structural integrity and functional activity. While the composition of commercial mitochondrial preservation solutions may vary depending on the brand or experimental requirements, they generally include the following core components: osmotic pressure regulators such as sucrose, antioxidants such as DTT (dithiothreitol) and β-mercaptoethanol, preservatives such as NaN3 (sodium azide), and stabilizers such as bovine serum albumin (BSA). However, some of these components are cytotoxic and cannot be used clinically. Furthermore, they lack GMP-compliant aseptic processing, posing a risk of microbial contamination. Therefore, current commercial mitochondrial preservation solutions cannot meet the requirements of clinical applications. Summary of the Invention

[0003] To solve the problems raised in the background art, the present invention provides a mitochondrial preservation composition and a preparation method thereof.

[0004] The technical solutions of the present invention are as follows: The invention provides a mitochondrial preservation composition, comprising 132 mM glucose, 85 mM sodium chloride and human serum albumin with a mass volume percentage concentration of 1%.

[0005] Furthermore, the mitochondrial preservation composition also includes sterile water for injection.

[0006] The physiological osmotic pressure of the mitochondria preservation composition is 280-320 mOsm / L.

[0007] The human serum albumin is injection grade human serum albumin.

[0008] The present invention also provides a method for preparing the mitochondrial preservation composition, comprising: The physiological osmotic pressure value is achieved by preparing 0.9% normal saline, 5% glucose solution, 3% NaCl solution and 20% human serum albumin; the total volume of the 0.9% normal saline and 3% NaCl solution and the volume ratio of the 5% glucose solution is 1:1.

[0009] Furthermore, 440 mL of 0.9% normal saline, 475 mL of 5% glucose solution, 35 mL of 3% NaCl solution, and 50 mL of 20% human serum albumin were prepared, and the total osmotic pressure of the liquid was 303.49 mOsm / L.

[0010] Beneficial effects The mitochondrial preservation composition obtained by the present invention through controlled proportions of commonly used clinical liquids: human serum albumin, glucose, and normal saline can maintain mitochondrial structure and functional activity, is suitable for in vivo transplantation and preservation, significantly reduces immunogenicity and toxicity, and meets clinical safety standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a comparison chart of the effects of different ratios of physiological saline and glucose solution on mitochondrial membrane potential.

[0012] Figure 2 3 is a comparison chart of the effects of the control group and the experimental group (the composition of the present invention) on mitochondrial membrane potential.

[0013] Figure 3 These are the test results of mitochondrial membrane potential of the control group and the experimental group (composition of the present invention) using the JC-1 kit.

[0014] Figure 4 The control group and the experimental group (the composition of the present invention) show the effects on the mitochondrial adenosine triphosphate (ATP) level.

[0015] Figure 5 This is a transmission electron microscope photograph of mitochondria after being stored in the storage solution of the present invention for 3 hours. DETAILED DESCRIPTION

[0016] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0017] The present invention provides a mitochondrial preservation composition, comprising 132 mM glucose, 85 mM sodium chloride, and human serum albumin with a mass volume percentage concentration of 1%.

[0018] The human serum albumin is injection-grade human serum albumin, which can provide colloidal osmotic pressure, stabilize mitochondrial structure, reduce immunogenicity, and protect mitochondrial membrane.

[0019] Furthermore, the mitochondrial preservation composition also includes sterile water for injection.

[0020] To ensure the structural integrity of mitochondria, preferably, the physiological osmotic pressure of the mitochondrial preservation composition should be 280-320 mOsm / L.

[0021] The present invention also provides a method for preparing the mitochondrial preservation composition, comprising: The physiological osmotic pressure value is achieved by preparing 0.9% normal saline, 5% glucose solution, 3% NaCl solution and 20% human serum albumin; the total volume of the 0.9% normal saline and 3% NaCl solution and the volume ratio of the 5% glucose solution is 1:1.

[0022] Preferably, the solution is prepared with 440 mL of 0.9% normal saline, 475 mL of 5% glucose solution, 35 mL of 3% NaCl solution and 50 mL of 20% human serum albumin, and the total osmotic pressure of the liquid is 303.49 mOsm / L.

[0023] The specific preparation process is as follows: Step 1: Determine the volumes of saline and glucose solutions Assume the final volume is 1 L (1000 mL), of which: 50 mL of human serum albumin (HSA) must be added, and the volumes of normal saline and glucose solutions must be reduced to a total of 950 mL.

[0024] Allocation in a 1:1 ratio: Normal saline: 475 mL; 5% glucose: 475 mL; 20% human serum albumin (HSA): 50 mL; Final volume: 475 + 475 + 50 = 1000 mL.

[0025] Step 2: Calculate the current osmotic pressure (1) NaCl concentration (from normal saline) Normal saline (0.9%) contains NaCl: 154 mM; 475 mL of normal saline → NaCl moles: 154 × 0.475 = 73.15 mmol; Final concentration (1L): 73.15 mM; Osmotic pressure contribution: 73.15×2=146.3 mOsm / L.

[0026] (2) Glucose concentration (from 5% glucose) 5% glucose solution contains glucose → molar concentration = (mass concentration) / (molar mass) = 50g / L / 180g / mol = 50 / 180mol / L = 5 / 18mol / L ≈ 0.2778 mol / L = 277.8 mM ≈ 278 mM; 475 mL 5% glucose → Moles of glucose: 278 × 0.475 = 132.05 mmol; Final concentration (1L): 132.05 mM; Osmotic pressure contribution: 132.05 mOsm / L.

[0027] Glucose is a non-electrolyte (i=1), osmotic concentration = 1×c glucose=1×0.13205mol / L=0.13205osm / L; Convert to mOsm / L: 0.13205 × 1000 = 132.05 mOsm / L.

[0028] (3) HSA osmotic pressure Negligible (<1 mOsm / L).

[0029] Current total osmotic pressure: 146.3 (NaCl) + 132.05 (glucose) = 278.35 mOsm / L; *(Below target 300 mOsm / L, need to replenish approximately 21.7 mOsm / L)* Step 3: Replenish osmotic pressure (replace part of the saline solution with 3% NaCl) Osmolality required: 21.7 mOsm / L; 3% NaCl (513 mM) osmolarity: 1026 mOsm / L (513 × 2); Therefore, 35 mL of 3% NaCl was added to replace the normal saline to replenish the osmotic pressure.

[0030] The adjusted composition formula is shown in Table 1.

[0031] Table 1 Adjusted composition formula Step 4: Verify final concentration and osmolarity (1) NaCl concentration From normal saline: 154 × 0.44 = 67.76 mmol; From 3% NaCl: 513×0.035=17.96mmol; Total NaCl: 67.76 + 17.96 = 85.72 mM; Osmolality: 85.72 × 2 = 171.44 mOsm / L.

[0032] (2) Glucose concentration From 5% glucose: 278 × 0.475 = 132.05 mmol → 132.05 mM; Osmolality: 132.05 mOsm / L.

[0033] (3) Total osmotic pressure 171.44+132.05=303.49mOsm / L.

[0034] *(Goal 300 mOsm / L)* The final composition formula is shown in Table 2.

[0035] Table 2 Final composition formula The mitochondrial preservation composition obtained by the present invention through controlled proportions of commonly used clinical liquids: human serum albumin, glucose, and normal saline can maintain mitochondrial structure and functional activity, is suitable for in vivo transplantation and preservation, significantly reduces immunogenicity and toxicity, and meets clinical safety standards.

[0036] Experiment 1: Verification of mitochondrial activity by different ratios of saline and glucose solutions method: Five groups of solutions with different ratios were prepared using 0.9% saline and 5% glucose solution as raw materials. The ratios of the two raw material solutions were 1:0 (100% NaCl), 0:1 (100% GLUCOSE), 1:3 (25% NaCl), 1:1 (50% NaCl) and 3:1 (75% NaCl), and the solutions were sterilized by filtration using a 0.22 μm filter membrane.

[0037] The isolated mitochondria were stored at 4°C using the five solutions prepared above in different proportions. After 3 hours, the mitochondrial red fluorescent probe (Mitotracker Red CMXRos) was used to quickly detect the activity of the mitochondria.

[0038] The average fluorescence intensity of Mitotracker Red CMXRos reflects mitochondrial activity. The higher the fluorescence intensity, the better the mitochondrial activity. Figure 1 ) showed that compared with other ratios, the 1:1 ratio of 0.9% saline to 5% glucose solution, that is, (50% NaCl), had the best preservation effect on mitochondria.

[0039] Experiment 2: Preparation of liquid to verify mitochondrial activity method: 1. Isolate mesenchymal stem cell mitochondria and divide them into two groups: Experimental group: the mitochondria preservation composition of the present invention was used as clinical fluid (stored at 4°C for 3 hours); Control group: commercial kit preservation solution (BestBio, BB-3612) was used (stored at 4°C for 3 hours).

[0040] 2. Detection indicators: Mitochondrial membrane potential was measured using Mitotracker Red CMXRos staining. Mitochondrial membrane potential is a core indicator for assessing mitochondrial function, reflecting electron transport chain activity and energy synthesis capacity. MitoTracker Red CMXRos is a cationic fluorescent dye whose accumulation is proportional to mitochondrial membrane potential and is widely used to measure mitochondrial membrane potential in living or fixed cells. Mitochondrial membrane potential was further measured using the JC-1 assay kit, which uses the red-to-green fluorescence ratio to reflect mitochondrial status; a higher ratio indicates greater activity.

[0041] Use the ATP detection kit, which uses the luciferase method to detect mitochondrial adenosine triphosphate (ATP) levels. ATP is a core molecule in cellular energy metabolism, and its level directly reflects the functional status of mitochondria. Observation of mitochondria by transmission electron microscopy directly reflects the structural integrity of mitochondria.

[0042] 3. Results By Mitotracker Red CMXRos ( Figure 2 ) and JC-1 detection kit ( Figure 3 ) Detection of mitochondrial membrane potential of the control group and experimental group, the results show that the difference in mitochondrial membrane potential after mitochondria were preserved in the experimental group and the control group preservation solution was small, and the mitochondrial membrane potential can be maintained during the preservation period. Figure 4 It can be seen that the ATP level was quantified by luminescence value (RLU). The mitochondria in the experimental group retained the ability to produce ATP after liquid storage, which protected the functional integrity of the mitochondria to a certain extent, which was in line with expectations. Figure 5 It can be seen that the mitochondria photographed after being preserved in the preservation solution of the invention for 3 hours were shown to be intact in morphology, with a typical oval shape, intact inner and outer membranes, and inner cristae with intact inner cristae structure. Therefore, the preservation solution of the invention can better protect the structural integrity of mitochondria.

Claims

1. A mitochondrial preservation composition, characterized in that The invention comprises 132 mM glucose, 85 mM sodium chloride, and human serum albumin with a mass volume percentage concentration of 1%.

2. The mitochondria preservation composition according to claim 1, characterized in that The mitochondria preservation composition further comprises sterile water for injection.

3. The mitochondria preservation composition according to claim 2, characterized in that The physiological osmotic pressure of the mitochondria preservation composition is 280-320 mOsm / L.

4. The mitochondria preservation composition according to claim 1, characterized in that The human serum albumin is injection grade human serum albumin.

5. A method for preparing the mitochondria preservation composition according to claim 2, characterized in that: include: The osmotic pressure was adjusted to 0.9% saline, 5% glucose solution, 3% NaCl solution and 20% human serum albumin. The volume ratio of the total volume of the 0.9% normal saline solution and the 3% NaCl solution to the volume of the 5% glucose solution is 1:

1.

6. The method for preparing the mitochondria-preserving composition according to claim 5, characterized in that: It is prepared with 440 mL of 0.9% normal saline, 475 mL of 5% glucose solution, 35 mL of 3% NaCl solution and 50 mL of 20% human serum albumin, and the total osmotic pressure of the liquid is 303.49 mOsm / L.

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