A method for preparing a primary hepatocyte preservation solution

Abstract

The application relates to the technical field of primary hepatocyte biological preservation, and discloses a preparation method of a primary hepatocyte preservation solution, which comprises the following steps: taking sterile deionized water, precooling the deionized water at 4 DEG C, and adding an inorganic salt component to prepare an electrolyte base solution; adding an osmotic regulator, an antioxidant and an energy substrate into the base solution; adding polyethylene glycol 8000 and stirring and dissolving; adjusting the pH value of the solution and filtering and sterilizing; the polyethylene glycol 8000 with a specific concentration is used to generate a volume exclusion effect in a low-temperature environment, a macromolecular crowded physical environment is constructed, the energy substrate is driven to be enriched to a damaged site of a hepatocyte membrane in a directional manner, then the diffusion resistance of small molecule components is offset, and micro cracks generated in a phase change process of a membrane lipid bilayer are repaired, in-situ repair of primary hepatocyte membrane damage is realized, and the cell survival rate is improved while the macroscopic osmotic pressure of the system is kept stable.

Description

Technical Field

[0001] This invention relates to the field of primary hepatocyte biological preservation technology, specifically to a method for preparing a primary hepatocyte preservation solution. Background Technology

[0002] Primary hepatocytes are currently the core material for liver physiology research and drug metabolism assessment. Maintaining their in vitro activity and function is a key prerequisite for related research and development. In current preservation processes, freshly isolated hepatocytes are usually placed in a 4°C environment for short-term cryopreservation. The preservation media used include UW solution, cell culture medium or specific ion buffer.

[0003] However, primary hepatocytes face clear physicochemical limitations under low-temperature conditions. When the temperature drops to 4°C, the lipid bilayer on the cell membrane surface undergoes a phase transition from a fluid to a gel state, resulting in phase transition cracks. This forms the pathological basis for false positives in trypan blue assays after cell damage. Simultaneously, low temperature causes a decrease in Brownian motion of molecules within the system, significantly reducing the diffusion coefficient and creating diffusion kinetic hindrance. In traditional preservation solutions, added energy substrates or repair components are homogeneously distributed throughout the solution. Due to the reduced diffusion rate, these components cannot reach the damage sites on the membrane surface in time before irreversible leakage and apoptosis occur. Besides adjusting the component ratios, existing technologies attempt to introduce combinations of biochemical protective agents to enhance cell tolerance, but... There is a fundamental mismatch between the design scheme for cryogenic conditions and the short-term temporary storage scenario at 4°C. For example, Chinese invention patent application CN110463689A discloses a primary hepatocyte cryopreservation solution, a hepatocyte cryopreservation method, and a hepatocyte resuscitation method. It constructs a cryogenic protection system using a high ratio of dimethyl sulfoxide (DMSO) and fetal bovine serum. The core technology lies in using a permeable protective agent to lower the intracellular freezing point. However, under the conventional non-freezing storage conditions at 4°C, there is a lack of physical constraints for ice crystal formation. Small molecule protective agents such as DMSO cannot solve the problem of the kinetics of the directional delivery of substrates to the damaged membrane interface. Furthermore, due to their high chemical activity and osmotic pressure overload in non-cryoprehy environments, they produce cytotoxicity to hepatocytes in a metabolically inhibited state, exacerbating the mechanical fragility and functional damage of the membrane structure.

[0004] Therefore, the technical problem to be solved by this invention is how to construct a microenvironment driven by the macromolecular volume exclusion effect, so as to achieve the directional enrichment and in-situ repair of energy repair substrates to the pericellular area of ​​hepatocytes while ensuring the osmotic pressure stability of the system. Summary of the Invention

[0005] To address the problems in the background art, this invention proposes a method for preparing a primary hepatocyte preservation solution, comprising the following steps: Step S101: Take sterile deionized water and pre-cool it at 4°C. Add sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and glucose. Adjust the mass of the electrolyte components so that the final concentration of sodium ions in the electrolyte base solution is 20 mmol / L to 30 mmol / L and the final concentration of potassium ions is 4 mmol / L to 6 mmol / L. Step S102: Raffinose, taurine, reduced glutathione, allopurinol, adenosine, and sodium pyruvate are added to the electrolyte base solution and stirred. The final concentration of raffinose is adjusted to 25 mmol / L to 35 mmol / L, the final concentration of reduced glutathione is adjusted to 2 mmol / L to 4 mmol / L, the final concentration of allopurinol is adjusted to 0.5 mmol / L to 1.0 mmol / L, the final concentration of adenosine is adjusted to 4 mmol / L to 6 mmol / L, and the final concentration of sodium pyruvate is adjusted to 1 mmol / L to 3 mmol / L. Step S103: Add polyethylene glycol 8000 to the electrolyte base solution and control the final concentration of polyethylene glycol 8000 in the obtained preservation solution to be 40 g / L to 60 g / L. Through the steric hindrance of the solvent by polyethylene glycol 8000, the diffusion distribution of reduced glutathione, allopurinol, adenosine and sodium pyruvate on the surface of primary hepatocyte membrane is constrained. In step S104, add sodium hydroxide solution or HEPES to adjust the pH of the solution to 7.0 to 7.4, and after making up the volume, filter through a 0.22μm sterile filter membrane.

[0006] Preferably, step S102 further includes the following sub-steps: step S1021, adding reduced glutathione with a mass concentration of 0.8 g / L to 1.2 g / L and allopurinol with a mass concentration of 0.12 g / L to 0.15 g / L to the electrolyte base solution, utilizing the space occupied by polyethylene glycol 8000 to make the concentration of reduced glutathione and allopurinol at the periphery of the primary hepatocyte membrane higher than their average concentration in the obtained preservation solution.

[0007] Preferably, the polyethylene glycol 8000 selected in step S103 is pharmaceutical grade, and its molecular weight distribution width index is less than or equal to 1.05.

[0008] Preferably, step S102 further includes the following sub-step: step S1022, adding adenine and ribose to the electrolyte base solution, wherein the amount of adenine added is 0.13 g / L to 0.14 g / L, and the amount of ribose added is 0.65 g / L to 0.70 g / L.

[0009] Preferably, in step S101, the initial osmotic pressure of the electrolyte base solution is controlled to be between 290 mOsm / L and 310 mOsm / L.

[0010] Preferably, step S104 further includes the following sub-steps: step S1041, placing the mixed solution in a constant temperature water bath and stirring at a speed of 100 rpm to 150 rpm for 20 min to 30 min at a temperature of 20°C to 25°C.

[0011] Preferably, the electrolyte base solution in step S101 is composed of sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, glucose, and sterile deionized water.

[0012] Preferably, step S102 further includes adding dexamethasone at a mass concentration of 0.05 g / L to 0.10 g / L to the electrolyte base solution.

[0013] Preferably, the preservation solution produced by the preparation method has a dynamic viscosity of no more than 3.5 mPa·s at 4°C, which is used to reduce shear damage when resuspending primary hepatocytes.

[0014] The beneficial effects of this invention are: 1. In the preparation of primary hepatocyte preservation solution, a physicochemical coupling of 50 g / L PEG8000 with adenosine and sodium pyruvate is used to establish a macromolecular crowding environment in a 4°C buffer system. The volume exclusion effect generated by this environment physically drives the migration of small molecule substrates to the lipid-water interface and phase transition microcracks of the hepatocyte membrane. This interface enrichment mechanism, which is constrained by the physical conformation of polymers, causes a nonlinear increase in the substrate concentration at the damaged membrane site. Without increasing the total osmotic pressure of the system, it overcomes the diffusion kinetic hindrance under low temperature conditions, providing sufficient substrate collision probability for the slow enzymatic repair process, and achieving in-situ repair of primary hepatocyte membrane damage. This is reflected in the increased cell viability after preservation compared to the initial preservation period.

[0015] 2. This scheme utilizes the volume exclusion mechanism to achieve substrate interface enrichment, resolving the systemic contradiction between the concentration of protective components and osmotic toxicity in the field of cell preservation. By physically occupying solvent space by macromolecules, while maintaining the total macroscopic osmotic pressure of the system within the physiological safety window, it changes the spatial distribution of energy supply substrates and antioxidant combinations, enabling the microenvironment of hepatocyte membranes to obtain repair kinetic energy far exceeding the macroscopic ratio concentration. This mechanism avoids the large fluctuations in osmotic pressure caused by increasing the total amount of substrate to pursue protective effects in traditional technologies, and enhances the energy supply efficiency at the microscopic level while ensuring the osmotic pressure homeostasis of hepatocytes.

[0016] 3. The low-sodium, low-potassium ion system, in synergy with PEG8000, creates a low-viscosity and ion-balanced preservation environment. The low-sodium system effectively inhibits calcium influx into cells at low temperatures, preventing apoptosis caused by calcium overload. The low-potassium design reduces the need for ion washing during resuscitation, minimizing secondary mechanical damage to weakened cell membranes caused by repeated centrifugation. Meanwhile, PEG8000 replaces high-viscosity macromolecular colloids, generating sufficient colloid osmotic pressure to inhibit cell swelling while ensuring the preservation solution possesses excellent rheological properties, ensuring uniform distribution of single-cell suspensions and improving the smoothness of hepatocyte resuspension and perfusion procedures. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart illustrating the standardized preparation process of the primary hepatocyte preservation solution of this invention. Figure 2 This is a logical architecture diagram of physical repair and biochemical metabolic compensation in the preparation of the preservation solution of this invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] A method for preparing a primary hepatocyte preservation solution includes the following steps: Step S101: Take sterile deionized water and pre-cool it at 4°C. Add sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and glucose. Adjust the mass of the electrolyte components so that the final concentration of sodium ions in the electrolyte base solution is 20 mmol / L to 30 mmol / L and the final concentration of potassium ions is 4 mmol / L to 6 mmol / L. Step S102: Raffinose, taurine, reduced glutathione, allopurinol, adenosine, and sodium pyruvate are added to the electrolyte base solution and stirred. The final concentration of raffinose is adjusted to 25 mmol / L to 35 mmol / L, the final concentration of reduced glutathione is adjusted to 2 mmol / L to 4 mmol / L, the final concentration of allopurinol is adjusted to 0.5 mmol / L to 1.0 mmol / L, the final concentration of adenosine is adjusted to 4 mmol / L to 6 mmol / L, and the final concentration of sodium pyruvate is adjusted to 1 mmol / L to 3 mmol / L. Step S103: Add polyethylene glycol 8000 to the electrolyte base solution and control the final concentration of polyethylene glycol 8000 in the obtained preservation solution to be 40 g / L to 60 g / L. Through the steric hindrance of the solvent by polyethylene glycol 8000, the diffusion distribution of reduced glutathione, allopurinol, adenosine and sodium pyruvate on the surface of primary hepatocyte membrane is constrained. In step S104, add sodium hydroxide solution or HEPES to adjust the pH of the solution to 7.0 to 7.4, and after making up the volume, filter through a 0.22μm sterile filter membrane.

[0021] Preferably, step S102 further includes the following sub-steps: step S1021, adding reduced glutathione with a mass concentration of 0.8 g / L to 1.2 g / L and allopurinol with a mass concentration of 0.12 g / L to 0.15 g / L to the electrolyte base solution, utilizing the space occupied by polyethylene glycol 8000 to make the concentration of reduced glutathione and allopurinol at the periphery of the primary hepatocyte membrane higher than their average concentration in the obtained preservation solution.

[0022] Preferably, the polyethylene glycol 8000 selected in step S103 is pharmaceutical grade, and its molecular weight distribution width index is less than or equal to 1.05.

[0023] Preferably, step S102 further includes the following sub-step: step S1022, adding adenine and ribose to the electrolyte base solution, wherein the amount of adenine added is 0.13 g / L to 0.14 g / L, and the amount of ribose added is 0.65 g / L to 0.70 g / L.

[0024] Preferably, in step S101, the initial osmotic pressure of the electrolyte base solution is controlled to be between 290 mOsm / L and 310 mOsm / L.

[0025] Preferably, step S104 further includes the following sub-steps: step S1041, placing the mixed solution in a constant temperature water bath and stirring at a speed of 100 rpm to 150 rpm for 20 min to 30 min at a temperature of 20°C to 25°C.

[0026] Preferably, the electrolyte base solution in step S101 is composed of sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, glucose, and sterile deionized water.

[0027] Preferably, step S102 further includes adding dexamethasone at a mass concentration of 0.05 g / L to 0.10 g / L to the electrolyte base solution.

[0028] Preferably, the preservation solution produced by the preparation method has a dynamic viscosity of no more than 3.5 mPa·s at 4°C, which is used to reduce shear damage when resuspending primary hepatocytes.

[0029] Example 1: In the application of ex vivo primary liver parenchymal cells for large-scale drug metabolism screening, when freshly isolated liver parenchymal single cells are placed under a low temperature condition of 4°C, the lipid bilayer on the cell membrane surface undergoes a phase transition from a fluid state to a gel state. During this phase transition, physical cracks at the molecular interstitial level are generated on the membrane surface due to changes in the arrangement of lipid molecules. At the same time, the 4°C environment weakens the Brownian motion of molecules in the system, and the diffusion coefficient of energy substrates and antioxidant components used to repair membrane damage in the fluid decreases, resulting in kinetic hindrance. This leads to a low concentration of repair substrates in the damaged area of ​​the membrane, making it difficult to complete in-situ repair before the cells undergo physical swelling.

[0030] The method for preparing primary hepatocyte preservation solution provided by this invention limits the final concentration of polyethylene glycol 8000 in the preservation solution to 50 g / L, and constructs a low-potassium and low-sodium ion buffer environment by combining it with 35 mmol / L potassium chloride and 30 mmol / L sodium chloride. At 4°C, this not only utilizes the high molecular colloidal properties of polyethylene glycol 8000 to generate a hyperosmolar anti-swelling effect in the overall system, but also establishes a critical macromolecular crowding physical environment in local spaces. In this physical environment, due to the steric hindrance of the long-chain polyethylene glycol 8000 molecules, it is difficult for it to enter the physical cracks on the surface of the hepatocyte membrane. Based on the volume exclusion effect, a thermodynamic driving force is generated, resulting in a homogeneous distribution of small molecule components within the system, namely 5 mmol / L adenosine, 3 mmol / L sodium pyruvate, and 3 mmol / L reduced form. Glutathione and 0.5 mmol / L allopurinol detach from the bulk solution and directionally accumulate at the lipid-water interface and damaged defects of the cell membrane, which cannot be occupied by polyethylene glycol molecules. Specifically, polyethylene glycol 8000 forms a huge hydration network in the aqueous phase and occupies a large amount of the effective solvent volume, which leads to a drastic compression of the available space for small molecules in the bulk solution. This sudden reduction in available space causes an abnormal increase in the macroscopic thermodynamic activity and chemical potential of small molecules in the bulk phase. In order to reduce the total free energy of the system, the substrate molecules spontaneously diffuse into the near-membrane microregions that polyethylene glycol cannot enter due to steric hindrance, based on the chemical potential gradient. This process does not rely on active cross-scale targeting guidance by vector force field, but on the spontaneous evolution of concentration redistribution based on exclusion thermodynamic equilibrium, which ultimately manifests as the passive dense accumulation of small molecules at the membrane interface.

[0031] This interface enrichment mechanism, driven by polymeric physical conformation, increases the effective collision probability of adenosine in the damaged membrane with repair substrates such as sodium pyruvate without increasing the total osmotic pressure of the preservation solution system. This allows for the anchoring of sufficient energy donors under a diffusion-blocked environment at 4°C, providing continuous kinetic energy for the reversible repair of the hepatocyte membrane lipid bilayer and achieving in-situ compensation for mechanical damage to isolated hepatocytes. Under the 4°C cryogenic inhibition state, the conventional mitochondrial oxidative phosphorylation pathway is almost halted due to limited enzyme activity. At this time, exogenous adenosine enriched around the membrane enters the cell down its concentration gradient through balanced nucleoside transport proteins that still maintain basal transport activity on the cell membrane surface. In the low-activation-energy-requirement nucleoside rescue pathway, adenosine, catalyzed by intracellular adenosine kinase, utilizes the cellular region... The remaining low-level substrate pool is directly converted into adenosine monophosphate (ATP), triggering bypass substrate-level phosphorylation to replenish the ATP pool. This mechanism completely bypasses the low-temperature thermodynamic barrier of the mitochondrial electron transport chain, supporting the minimum energy consumption required for lipid turnover and structural remodeling of damaged membranes through a biochemical compensatory pathway. Experimental results show that after the preservation solution produced by this preparation method is stored at 4°C for 10 hours, the trypan blue rejection rate of primary hepatocytes shows a trend of increasing by 5% to 15% compared with the initial isolation. The cell morphology remains round and the edges are smooth, and the fragmentation rate is maintained below 5%. Moreover, the albumin secretion and cytochrome P450 enzyme activity retention rate after recovery are both greater than or equal to 70%, confirming that the physical dimension of substrate-directed delivery means and the biochemical dimension of energy compensation mechanism produce a synergistic effect.

[0032] Example 2: In the cold chain handling application of isolated primary hepatocytes for large-scale drug metabolism kinetic screening, a biological experimental platform with temperature control precision maintained at 0.1°C and 50Hz mechanical oscillation simulation function was used to verify the intracellular enzyme leakage and apoptosis challenge caused by lipid bilayer phase transition cracks during temporary storage of freshly isolated primary hepatocytes at 4°C. When preparing the preservation solution, the initial pH was set to 7.42 because the phosphate buffer component exhibits proton release at 4°C. Regarding the selection of the polyethylene glycol 8000 concentration, a balance was struck between the physical driving force generated by the exclusion volume and the fluid viscosity caused by the increase in polymer concentration. When the concentration was set to 50 g / L, the exclusion volume of macromolecules within the system reached a critical overlap state, generating a chemical reaction that caused small molecule substrates to migrate to the membrane interface. The measured dynamic viscosity of the system was 1.32 mPa·s, which maintained physical safety during cell resuspension. The critical overlap concentration was determined based on the Mark-Houwink equation. In an aqueous solution at 4°C, the intrinsic viscosity of the selected polyethylene glycol 8000 was calibrated to be 18.6 to 20.2 mL / g. According to the theory of dilute solutions in polymer physics, the critical overlap concentration is approximately equal to the reciprocal of the intrinsic viscosity. The calculated theoretical value is in the range of 49.5 g / L to 53.7 g / L. Therefore, setting the lower limit of the concentration to 40 g / L can ensure that the system is close to the edge of the overlap state to initiate the exclusion effect, while setting the upper limit to 60 g / L can prevent the polymer chains from becoming completely entangled, which would cause an exponential jump in fluid viscosity. 50 g / L is exactly the middle value of the safe transition window verified by this physical derivation model.

[0033] Multiple control systems were set up to elucidate the interaction mechanism among the components. The experimental group used a complete preservation solution prepared by the method of this invention, containing 50 g / L polyethylene glycol 8000, 5 mmol / L adenosine, and 3 mmol / L sodium pyruvate. Control group one used a buffer solution without polyethylene glycol 8000 but containing an equal amount of energy substrate; control group two used a solution containing 50 g / L polyethylene glycol 8000 but without adenosine and sodium pyruvate; and control group three used a low-concentration solution of 30 g / L polyethylene glycol 8000. After preservation for 10 hours at 4°C with mechanical vibration at a noise level of 20 dB Gauss, the trypan blue rejection rate was detected by flow cytometry. The results showed that control group one had a viability of 52.4% because the substrate could not reach the damaged sites due to low-temperature diffusion hindrance; control group two lacked biochemical repair kinetics and had a viability of 61.8%; and control group three had enrichment due to the exclusion volume not reaching the critical threshold. The efficiency was insufficient, with a survival rate of 68.3%, while the experimental group achieved a survival rate of 88.6%, showing a non-linear performance leap. This confirms the synergistic effect between the physical driving architecture and the biochemical repair pathway. To confirm the high concentration enrichment of repair substrates at the membrane periphery in a macromolecular crowded field, the phase distribution of the mixture was physicochemically verified using high-performance liquid chromatography. By introducing simulated cell membrane liposomes composed of a phospholipid bilayer into the preparation system and centrifuging them after static partitioning, the mixture was separated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. The results showed that the concentration of reduced glutathione in the supernatant obtained by ultrafiltration was significantly reduced. Furthermore, by eluting and quantifying the hydration layer attached to the liposome filter cake interface, it was confirmed that more than 60% of the small molecule components in the system were pushed away from the bulk phase by physical exclusion and tightly adhered to and enriched in the hydration layer space at the periphery of the liposome, clearly verifying the objective existence of heterogeneous concentration at the interface.

[0034] In a gradient optimization experiment targeting the sodium to potassium ion concentration ratio in an electrolyte base solution, to address the contradiction between cell membrane resting potential depolarization caused by a high potassium environment and delayed cell swelling induced by sodium ion overload, a parameter space of 20 mmol / L to 30 mmol / L sodium ion final concentration and 4 mmol / L to 6 mmol / L potassium ion final concentration was systematically selected for efficacy verification. The experiment observed that when the final potassium ion concentration was below the lower limit of 4 mmol / L, the excessive intracellular and extracellular ion gradient led to increased potassium ion efflux, inducing early apoptosis signal transduction. However, when the final potassium ion concentration exceeded the upper limit of 6 mmol / L, due to transmembrane potential fluctuations, the cell's tolerance to low temperature decreased, with the measured retention rate of cytochrome P450 enzyme activity decreasing from 75.8% to 58.2% after 10 hours. Correspondingly, when the final sodium ion concentration was maintained at the median point of 25 mmol / L, the macroscopic effect of combining 50 g / L polyethylene glycol 8000... Osmotic pressure compensation maintained the cell volume change rate within a steady-state range of ±3%. Compared to the out-of-range control group with sodium ion concentrations exceeding 30 mmol / L, the survival rate after resuscitation increased by 12.4%, confirming the physicochemical supporting role of this electrolyte range in maintaining transmembrane ion homeostasis in primary hepatocytes. At the underlying physicochemical mechanism, a 4°C cold environment inhibits the enzymatic activity of the sodium-potassium pump on the hepatocyte membrane, preventing the normal expulsion of intracellular sodium ions. If a conventional high-sodium preservation solution is used, the originally extremely high transmembrane sodium concentration gradient of the cells will disappear or even reverse, causing the sodium-calcium exchangers on the membrane surface, which depend on this gradient, to enter a reverse working mode, thereby pumping a large amount of extracellular calcium ions into the cell. This system actively reduces the extracellular sodium potential by setting a low sodium external environment of 20 mmol / L to 30 mmol / L, blocking the initiation conditions for the reverse operation of sodium-calcium exchangers from the physical source, avoiding the activation of the reverse calcium pumping mechanism, and achieving substantial inhibition of abnormal calcium influx at low temperature.

[0035] Example 3: This example combines Figures 1 to 2 A method for preparing a primary hepatocyte preservation solution is described, such as... Figure 1As shown, in step S101, sterile deionized water is pre-cooled at 4°C, and sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, and glucose are added to adjust the electrolyte composition so that the final sodium ion concentration in the electrolyte base solution is 20 mmol / L to 30 mmol / L and the final potassium ion concentration is 4 mmol / L to 6 mmol / L. Then, in step S102, raffinose, taurine, reduced glutathione, allopurinol, adenosine, and sodium pyruvate are added to the electrolyte base solution, and the mixture is stirred to adjust the final raffinose concentration to 25 mmol / L to 35 mmol / L, the final reduced glutathione concentration to 2 mmol / L to 4 mmol / L, and the final allopurinol concentration to... The adenosine concentration was set at 0.5 mmol / L to 1.0 mmol / L, the sodium pyruvate concentration was set at 4 mmol / L to 6 mmol / L, and the sodium pyruvate concentration was set at 1 mmol / L to 3 mmol / L. Then, in step S103, polyethylene glycol 8000 was added to the electrolyte base solution to control the final concentration of polyethylene glycol 8000 in the resulting preservation solution to 40 g / L to 60 g / L. The steric hindrance effect was used to restrict the diffusion and distribution of reduced glutathione, allopurinol, adenosine, and sodium pyruvate on the surface of the primary hepatocyte membrane. Finally, in step S104, sodium hydroxide solution or HEPES was added to adjust the pH of the solution to 7.0 to 7.4 and the solution was brought to a final volume and then filtered through a 0.22 μm sterile filter membrane.

[0036] like Figure 2 As shown, the logical architecture of the standardized preparation decision core of primary hepatocyte preservation solution is illustrated. The first end is the primary hepatocyte preservation solution standardized preparation decision core node, which points downwards to the environmental parameter determination node. This determination node points to the right to the environmental control node, which includes a 4°C pre-cooling environment, pH 7.0 to 7.4, and 0.22μm sterile filtration conditions. It also branches downwards into two decision nodes: ion balance decision and physical repair decision. The ion balance decision node points downwards to the sodium ion system (20 mmol / L to 30 mmol / L) node for inhibiting low-temperature calcium influx and the potassium ion system (4 mmol / L). The nodes maintain transmembrane ion homeostasis at concentrations from 1 / L to 6 mmol / L, and the physical repair decision nodes point downwards to polyethylene glycol 8000 at concentrations from 40 g / L to 60 g / L, respectively. These nodes construct a macromolecular crowding environment and a volume exclusion effect-driven substrate-directed enrichment node for in-situ repair of membrane damage. The sodium ion system node, potassium ion system node, and polyethylene glycol 8000 node converge downwards to the biochemical metabolic compensation node, which ultimately points to the final output configuration node containing adenosine 4-6 mmol / L, sodium pyruvate 1-3 mmol / L, and reduced glutathione 2-4 mmol / L.

[0037] Example 4: In large-scale primary hepatocyte preparation and cold chain distribution applications for preclinical toxicology evaluation, when preparing preservation solutions with a total batch volume exceeding 10L, if a simultaneous component addition dissolution method is used, the polyethylene glycol 8000 polymer chains exhibit water absorption and swelling properties in the aqueous phase, leading to the formation of dense hydrated micelles that encapsulate undissolved adenosine and reduced glutathione. This results in heterogeneous particles, causing crystallization during temporary storage at 4°C due to localized solubility product excess, disrupting the osmotic pressure balance of the system and causing mechanical damage to the cell membrane. The preparation process involves establishing a mixed flow field using a magnetically driven circulating pump under 4°C circulating cooling. Sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added sequentially to sterile deionized water until completely dissolved to construct the electrolyte framework. Following this, [further steps are taken]. Adenosine (5 mmol / L), sodium pyruvate (3 mmol / L), and reduced glutathione (3 mmol / L) were added dropwise to a final concentration. The ionic strength fluctuations of the solution were monitored in real time using an online conductivity sensor. Once the conductivity reading stabilized within ±0.05 mS / cm, polyethylene glycol 8000 was slowly added while adjusting the rotation speed to 350 r / min to maintain the tangential shear force of the system, ensuring that the long polymer chains were uniformly distributed around the formed ionic cloud during the unfolding process. In this preparation pathway, the technique of first establishing a small molecule dissolution equilibrium and then introducing a large molecule crowding field avoids the uneven solute distribution caused by polymer micelle encapsulation. Furthermore, the diffusion uniformity of the small molecule components was determined by monitoring the slope change of the conductivity online, transforming the dissolution endpoint into a process control node based on the evolution of physical quantities.

[0038] In preparing a composite protective system containing raffinose and taurine, to address the bottleneck of decreased cell membrane phospholipid head packing density and membrane lipid peroxidation caused by water molecule infiltration under low-temperature conditions, the preparation procedure limited the final concentration of raffinose to 25 mmol / L to 35 mmol / L, and combined it with 2 mmol / L to 4 mmol / L of taurine to construct a chemical potential shielding layer. Raffinose, as a high-molecular-weight non-reducing trisaccharide, replaces the hydration layer lost due to cooling by forming a hydrogen bond network with polar groups on the cell membrane surface, generating a steric hindrance effect to prevent the disordered aggregation of water molecules at membrane cracks. Taurine, as a sulfur-containing amino acid derivative, maintains the structural integrity of the lipid bilayer by quenching the free radical chain reaction generated by mechanical oscillation. When the raffinose concentration is below 25 mmol / L, the hydrogen bond support points on the membrane surface are insufficient, causing the cell fragmentation rate during resuspension to drop from 4.8%. The concentration increased to 13.5%, and when it exceeded 35 mmol / L, significant intracellular dehydration was observed due to the deviation of the system's chemical potential from the physiological window, with a measured decrease in albumin secretion of 21.6%. The experimental results showed that within this specific ratio range, the spatial shielding effect of raffinose and the free radical scavenging mechanism of taurine produced synergistic protection at the physical level, achieving deep homeostasis regulation of the primary hepatocyte membrane microenvironment. Experimental data showed that after 30 days of sealed storage at 4°C in the dark, the 10L preservation solution prepared by the above process maintained a transmittance of over 99% and no microcrystal precipitation. After 10 hours of storage, the albumin secretion of primary hepatocytes was 46.2 μg / d, and the cytochrome P450 enzyme activity retention rate was 75.8%. The coefficient of variation between batches was less than 3.5%, confirming the physical effectiveness of the preparation procedure in ensuring the stability of the macromolecular crowded environment and realizing the process conversion from formulation to large-scale preparation.

[0039] Example 5: In the standardized preparation of primary hepatocyte preservation solution involving different batches of raw material composition, the degree of polymerization of polyethylene glycol 8000 from different sources varies, directly causing fluctuations in its chain segment unfolding rate and the shielding effect of electrolyte clouds in the aqueous phase. Therefore, before starting the dropping program, a process parameter calibration procedure based on the change in conductivity slope needs to be executed according to the characteristics of the current batch of raw materials. This procedure selects 100 mL of electrolyte base solution as the calibration sample, and slowly drops the polyethylene glycol 8000 mother liquor at a fixed flow rate of 5 mL per minute under constant temperature cycling conditions at 4°C. The ion strength data output by the online conductivity sensor is collected in real time, and the rate of change of conductivity with the dropping volume is calculated using a data processor. The inflection point of this rate of change is used to identify the saturation moment of polymer occupying vacancies. When the rate of change of conductivity at 5 consecutive sampling points is less than 0.01 mS / (c When the conductivity reading is m·mL, the system sets the conductivity reading under this state as the drop addition termination reference value for this batch of preparation, thereby transforming the dissolution uniformity into a controlled physical measurement index and eliminating the disturbance of material batch differences on the construction process of the macromolecular crowded environment. The monitoring mechanism is that although polyethylene glycol, as a non-ionic polymer, does not release ions, its polymer chain segments continuously combine with free water molecules to build a dense hydration shell during the unfolding process. This physical water-taking process reduces the volume of free water acting as a solvent, causing the original sodium, potassium and other electrolyte ions in the system to be passively concentrated. Macroscopically, this is manifested as a linear increase in solution conductivity with the drop addition process. When the inflection point where the rate of change of conductivity approaches zero appears, it means that the free water in the system has been taken up to the dynamic limit boundary by the polymer hydration process, and the effective space occupancy has reached a saturated state. Thus, the macroscopic ionic conductivity characteristics are used to accurately map the microscopic physical conformation evolution of polymer chain segments in the liquid phase.

[0040] When the system faces changes in hydrodynamic state caused by fluctuations in ambient temperature, a compensation procedure for the rotational speed of the magnetically driven circulating pump needs to be executed to maintain the consistency of the tangential shear distribution of polyethylene glycol 8000 long-chain molecules in the solution. The preparation system uses a built-in temperature sensor to acquire the fluid temperature in the mixing chamber in real time, and according to the preset fluid viscosity-temperature characteristic function, when the temperature deviates from the 4°C reference point by more than 0.2°C, the rotational speed of the driving pump is increased by 10 r / min for every 0.1°C increase, in order to offset the effect of increased polymer thermal motion caused by temperature rise on the stability of the volume exclusion field. After such closed-loop adjustment and pre-calibration of physical parameters, even within a reasonable tolerance range of less than 5% in the raw material specifications, the prepared preservation solution still maintains a constant molecular-level osmotic pressure gradient. The preservation solution produced under this calibration procedure shows batch-to-batch stability in its protective efficacy against primary hepatocytes, and the fluctuation range of trypan blue rejection rate after 10 hours of preservation narrows to less than 2%, confirming the logical correspondence between process control nodes and material characteristics.

[0041] Example 6: In the batch validation scenario of primary hepatocyte preservation solution for automated continuous preparation line operation, due to physical differences in environmental electromagnetic intensity and initial water ion abundance between production workshops in different geographical locations, the system adopts a set of on-site benchmark calibration procedures for online conductivity sensors and high-precision pH meters to maintain the accuracy of macromolecular crowding field construction. This procedure selects a 100 mmol / L potassium chloride standard solution prepared with sterile deionized water as the conductivity calibration benchmark. In a closed constant-temperature mixing tank at 4°C, the gain coefficient of the conductivity sensor is adjusted to ensure that the deviation between the measured reading and the standard value is less than 0.01 mS / cm. Based on this, a three-point calibration method is implemented for the pH meter, using standard buffer solutions with pH values ​​of 4.01, 7.00, and 9.21 sequentially for slope compensation. The temperature during the compensation process is also controlled. The temperature sensor feedback value is locked in the range of 4.0°C to 4.2°C, thereby eliminating the interference of low temperature environment on ion activity detection and providing a physical basis for step-by-step addition of energy substrate. In actual production, the use of a single-component potassium chloride solution as the initial reference is not an attempt to simulate the real chemical environment of complex multi-component protective liquid, but to perform basic physical zeroing of the hardware polarization deviation of the online conductivity sensor. This is because the cell constant of the sensor is very prone to hardware drift under continuous mechanical scouring of cold fluid at 4°C. The introduction of a general strong electrolyte standard solution can force the gain slope of the probe back to the standard linear working range. After completing the pure physical level hardware constant correction, the probe can accurately and sensitively capture the relative conductivity change trend triggered by the subsequent addition of complex components, cutting off the influence of the instrument's inherent drift on the subsequent concentration algorithm judgment.

[0042] When the system enters the sterile filling mode, the viscosity of polyethylene glycol 8000 (50 g / L) increases. To avoid pressure overload causing shear damage to the long polymer chains, the system implements a segmented pressure control and flow feedback filtration procedure. The preparation system introduces the uniformly mixed solution into a 0.22-micron polyethersulfone filter array with an effective filtration area of ​​0.5 square meters. By adjusting the output power of the diaphragm pump, the pre-filtration pressure is maintained within a constant range of 0.15 MPa to 0.18 MPa, and the post-filtration flow rate is monitored in real time. If the flow rate decline slope exceeds a preset threshold of 10 mL per minute, a low-frequency pulse backflushing action is triggered to remove the polymer polarization layer on the filter membrane surface. Experimental data shows that the preservation solution treated by this procedure maintains a viscosity of 4... After standing at °C for 24 hours, the molecular weight distribution curve of polyethylene glycol 8000, as determined by gel permeation chromatography, remained consistent with that of the original material. No characteristic peaks of long-chain fragments were observed, and the sterility test of the product was negative. To maintain the conformational stability of the polymer under mass production conditions, sodium chloride and potassium chloride were added to pre-cooled sterile deionized water at 4°C during the preparation of the electrolyte base solution. The real-time ion activity of the mixture was monitored using an online ion-selective electrode, and the feed pump speed was adjusted accordingly to ensure that the final sodium ion concentration in the mixture was between 20 mmol / L and 30 mmol / L, and the final potassium ion concentration was between 4 mmol / L and 6 mmol / L. To prepare the small molecule protection system, raffinose and reduced glutathione were added to the mixture and added dropwise simultaneously. Taurine was added, and the final concentration was controlled within the range of 2 mmol / L to 4 mmol / L. After the initial dispersion of polyethylene glycol 8000, the solution was placed in a constant temperature water bath and heated to 20°C to 25°C while stirring at 100 rpm to 150 rpm for 20 to 30 minutes. The heating allowed the polyethylene glycol 8000 polymer chains to expand and dehydrate in the aqueous phase, eliminating the local high-viscosity aggregates caused by the macromolecular network. The fluid state met the rheological conditions for subsequent pore size sieving. The system was introduced into polyethylene glycol 8000, and the endpoint was determined by the rate of change in conductivity. A feed pump was used to inject the mother liquor at a flow rate of 5 mL / min. An online conductivity sensor collected flow field data at a sampling frequency of 1 Hz. The processor calculated the rate of change of conductivity with volume based on data from adjacent sampling points. When the rate of change is less than 0.01 mS / cm·mL for 10 consecutive sampling cycles, the feeding pipeline is cut off. When the concentration is in the range of 40 g / L to 60 g / L, the mixture is introduced into the filtration process after being heated in a water bath. The mixture is then introduced into a 0.45 μm polyethersulfone pre-filtration membrane to trap undissociated polymeric hydrated micelles. The pre-treated solution enters a 0.22 μm sterile filter array. The power output of the diaphragm pump is adjusted to control the pressure at the pre-filtration monitoring point between 0.15 MPa and 0.18 MPa. When the flow meter feedback decay rate is greater than 10 mL / min, the back pressure cleaning program is triggered. A single pulse backflow airflow lasting 0.8 seconds with a reverse pressure difference of 0.05 MPa is applied to strip away the residual surface polarization layer. The cold chain storage is then terminated, and the recovery process is performed using a solution containing 0.The cell suspension was diluted by equal volume with 1% bovine serum albumin and physiological saline. Injection of physiological saline disrupted the continuous size exclusion network, reducing the dynamic viscosity of the liquid phase to below 1.0 mPa·s, restoring the Newtonian fluid properties of the cell suspension. This rheological change eliminated liquid adhesion to the cell walls during pipetting or microfluidic perfusion operations. Cell counting deviation within the multi-well plate was controlled within 3%, meeting the homogeneity requirements of high-throughput drug screening platforms for single-cell suspensions.

[0043] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a primary hepatocyte preservation solution, characterized in that, Includes the following steps: Step S101: Take sterile deionized water and pre-cool it at 4°C. Add sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and glucose. Adjust the mass of the electrolyte components so that the final concentration of sodium ions in the electrolyte base solution is 20 mmol / L to 30 mmol / L and the final concentration of potassium ions is 4 mmol / L to 6 mmol / L. Step S102: Raffinose, taurine, reduced glutathione, allopurinol, adenosine, and sodium pyruvate are added to the electrolyte base solution and stirred. The final concentration of raffinose is adjusted to 25 mmol / L to 35 mmol / L, the final concentration of reduced glutathione is adjusted to 2 mmol / L to 4 mmol / L, the final concentration of allopurinol is adjusted to 0.5 mmol / L to 1.0 mmol / L, the final concentration of adenosine is adjusted to 4 mmol / L to 6 mmol / L, and the final concentration of sodium pyruvate is adjusted to 1 mmol / L to 3 mmol / L. Step S103: Add polyethylene glycol 8000 to the electrolyte base solution and control the final concentration of polyethylene glycol 8000 in the obtained preservation solution to be 40 g / L to 60 g / L. Through the steric hindrance of the solvent by polyethylene glycol 8000, the diffusion distribution of reduced glutathione, allopurinol, adenosine and sodium pyruvate on the surface of primary hepatocyte membrane is constrained. In step S104, add sodium hydroxide solution or HEPES to adjust the pH of the solution to 7.0 to 7.4, and after making up the volume, filter through a 0.22μm sterile filter membrane.

2. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, Step S102 further includes the following sub-steps: Step S1021, adding reduced glutathione with a mass concentration of 0.8 g / L to 1.2 g / L and allopurinol with a mass concentration of 0.12 g / L to 0.15 g / L to the electrolyte base solution, and using the space occupied by polyethylene glycol 8000 in the solvent, so that the concentration of reduced glutathione and allopurinol at the periphery of the primary hepatocyte membrane is higher than their average concentration in the obtained preservation solution.

3. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, The polyethylene glycol 8000 selected in step S103 is of pharmaceutical grade and its molecular weight distribution width index is less than or equal to 1.

05.

4. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, Step S102 further includes the following sub-steps: Step S1022, adding adenine and ribose to the electrolyte base solution, wherein the amount of adenine added is 0.13 g / L to 0.14 g / L, and the amount of ribose added is 0.65 g / L to 0.70 g / L.

5. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, In step S101, the initial osmotic pressure of the electrolyte base solution is controlled to be between 290 mOsm / L and 310 mOsm / L.

6. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, Step S104 further includes the following sub-steps: Step S1041, the mixed solution is placed in a constant temperature water bath and stirred at 100 rpm to 150 rpm for 20 min to 30 min at 20°C to 25°C.

7. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, In step S101, the electrolyte base solution consists of sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, glucose, and sterile deionized water.

8. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, In step S102, dexamethasone with a mass concentration of 0.05 g / L to 0.10 g / L is added to the electrolyte base solution.

9. The method for preparing a primary hepatocyte preservation solution according to claim 1, characterized in that, The preservation solution produced by the preparation method has a dynamic viscosity of no more than 3.5 mPa·s at 4°C, which can be used to reduce shear damage when resuspending primary hepatocytes.

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