A perfusate preparation process for efficiently preparing rodent primary hepatocytes

Abstract

The present application relates to the technical field of primary hepatocyte preparation, and discloses a perfusion fluid preparation process for efficiently preparing rodent primary hepatocytes, which comprises: pumping preheated (to 37 DEG C) pre-perfusion fluid containing ascorbic acid and chelating agent into the liver to stabilize the cell membrane and loosen cell connections; using 37 DEG C buffer to replace residual substances; then introducing preheated (to 37 DEG C) digestion fluid containing hyaluronidase and collagenase P and adjusting the flow rate to half of that in the pre-perfusion stage, so as to degrade the matrix and construct deep penetration channels; and finally, terminating the digestion under the condition of 4 DEG C. The present application cooperatively constructs a cell membrane stabilization environment to inhibit stress damage in the initial perfusion stage, realizes ion environment replacement, guarantees the uniformity of subsequent enzymolysis kinetics, further constructs a hierarchical penetration enzymolysis path, solves the uneven tissue dissociation contradiction, preserves the receptor activity and improves the output efficiency.

Description

Technical Field

[0001] This invention belongs to the field of primary hepatocyte preparation technology, and particularly relates to a process for preparing perfusion fluid for the efficient preparation of primary hepatocytes of rodents. Background Technology

[0002] Obtaining highly active and high-yield primary rodent hepatocytes is the foundation for conducting in vitro experiments. Currently, the classic two-step perfusion method is commonly used in this field, which involves perfusing calcium-free pre-perfusion fluid with calcium-containing collagenase digestion fluid, using chelating agents to detach intercellular junctions and relying on collagenase to degrade the extracellular matrix.

[0003] Because hepatocytes are highly sensitive to the mechanical shearing forces and microenvironmental osmotic pressure fluctuations generated by perfusion after being removed from the in situ circulatory system, existing technical solutions focus on the accumulation of chemical components, neglecting the protection of cell membrane potential stability in the early stages of perfusion. During the blood washing and decalcification stages, the rapid expansion of intercellular spaces is accompanied by oxidative stress in the cell membrane phospholipid layer, leading to irreversible submicroscopic damage to hepatocytes before entering the digestion stage. This logic of damage before dissolution limits the final survival rate of primary cells. For example, Chinese invention patent CN102634480B discloses a method for isolating and culturing primary liver cells, which standardizes the solution formulation and centrifugation purification steps. However, under actual working conditions, the pre-perfusion stage fails to construct a biochemical buffer barrier against reactive oxygen species induced by mechanical shearing. At the same time, global digestion by a single component of collagenase is difficult to penetrate the dense osmotic barrier composed of glycosaminoglycans inside the liver lobules, causing spatial unevenness contradictions such as over-digestion at the edges and insufficient dissociation in the core. Blindly prolonging the perfusion time will reach the physiological pressure threshold of cells, resulting in impaired cell surface receptor activity.

[0004] Therefore, the technical problem to be solved by this invention is how to establish a perfusion process that takes into account both cell membrane prestress protection and hierarchical permeation and enzymatic hydrolysis, so as to achieve uniform dissociation of hepatocytes and preserve the activity of cell surface receptors. Summary of the Invention

[0005] The present invention aims to solve the problems of low cell activity and unstable yield of hepatocytes due to oxidative stress damage and insufficient enzymatic digestion during perfusion dissociation.

[0006] In this technical solution, a process for preparing perfusion fluid for the efficient preparation of primary rodent hepatocytes includes the following steps: Step S101: Pre-perfusion fluid preheated to 37°C and with a pH of 7.4 is pumped into the isolated liver of a rodent. The pumping flow rate is set to the first flow rate and is maintained for 2 minutes. The pre-perfusion fluid contains 0.92 g / L ascorbic acid, 0.5 g / L HEPES, 0.19 g / L EDTA, and DPBS buffer. Ascorbic acid removes reactive oxygen free radicals in the interstitial spaces of the liver tissue under the mechanical shearing environment generated by the first flow rate, and works with EDTA to chelate intercellular calcium ions to loosen the hepatocyte connections. Step S102: Pump DPBS buffer at 37°C into the liver, maintaining the first flow rate for 1 minute, and replacing residual EDTA and metabolites in the liver tissue with DPBS buffer. Step S103: Preheated digestive fluid to 37°C is pumped into the liver for stratified dissociation. The pumping flow rate is set to the second flow rate and is maintained for 3 minutes. The digestive fluid contains 10 mg / L hyaluronidase, 200 mg / L collagenase P, and basal culture medium. The second flow rate is 0.5 times that of the first flow rate. Hyaluronidase degrades glycosaminoglycans in the liver tissue matrix to establish a penetration channel for collagenase P into the deep layers of the liver lobules. Step S104: Immerse the liver tissue after hierarchical dissociation in a stop solution at 4°C. The stop solution contains 5% fetal bovine serum and 1% penicillin / streptomycin mixture by volume.

[0007] Preferably, in step S101, the concentration of EDTA in the pre-perfusion fluid is 0.5 mmol / L.

[0008] Preferably, the concentration of ascorbic acid in the pre-perfusion fluid is from 0.1 mmol / L to 0.5 mmol / L.

[0009] Preferably, in step S101, the DPBS buffer contains NaCl at a concentration of 130 mmol / L to 145 mmol / L and [unclear - possibly a specific ingredient or concentration] at a concentration of 5.0 mmol / L to 6.0 mmol / L. And concentrations of 1.0 mmol / L to 1.5 mmol / L .

[0010] Preferably, in step S103, the digestive fluid is pre-treated with 0.22... Microporous membrane filtration for sterilization.

[0011] Preferably, in step S103, the basal culture medium is DMEM culture medium.

[0012] Preferably, the pH of the termination solution is 7.3 to 7.5.

[0013] Preferably, after step S104, the method further includes: step S105, peeling off the liver capsule with ophthalmic forceps and releasing hepatocytes into the termination solution with mechanical vibration; step S106, filtering the termination solution through a 100-mesh nylon filter, collecting the filtrate and centrifuging it at 4°C, with the centrifugation speed set to 50g to 100g and the centrifugation time to 2min to 5min.

[0014] Preferably, after centrifugation, the cell pellet is resuspended in a washing solution and centrifuged 2 to 3 times to ensure that the viability of the obtained rodent primary hepatocytes is not less than 95%.

[0015] Compared with existing technologies, the present invention provides a highly efficient process for preparing perfusion fluid for primary rodent hepatocytes, which has the following advantages: 1. In the efficient preparation of perfusion fluid for primary rodent hepatocytes, a stable cell membrane environment is synergistically constructed to inhibit stress damage in the early stage of perfusion. In the pre-perfusion stage, a microenvironment with redox buffering capacity is constructed by introducing a high concentration of ascorbic acid into a buffer system containing chelating agents. When the chelating agent strips calcium ions from the cells, causing the connection to loosen, ascorbic acid rapidly captures reactive oxygen free radicals induced by mechanical shearing force, maintains the physicochemical stability of the phospholipid layer of the hepatocyte membrane, avoids irreversible submicroscopic structural damage to hepatocytes before entering the enzymatic digestion stage, and ensures that the cells are in a highly active protective state from the initial stage of in vitro perfusion.

[0016] 2. Achieving precise replacement of the ionic environment ensures the uniformity of subsequent enzymatic hydrolysis kinetics. By setting up an in-situ flushing step with a single-component buffer between the pre-perfusion and digestion steps, residual chelating agents and metabolic byproducts in the interstitial space are effectively removed. This eliminates the potential inhibition of the chelating agent on the metal ion-dependent activity of collagenase, creating constant ionic strength and pH conditions for the binding and catalysis of collagenase at the liver tissue interface, thereby improving the initiation speed of the enzymatic hydrolysis reaction and the release stability between batches.

[0017] 3. A hierarchical enzymatic hydrolysis pathway is constructed to resolve the spatial inhomogeneity of tissue dissociation. A composite enzymatic hydrolysis system composed of hyaluronidase and collagenase P is adopted, combined with asymmetric flow rate control that is halved compared to the pre-perfusion stage, to form a deep dissociation mode from the inside out. Hyaluronidase first degrades glycosaminoglycans in the matrix and opens up molecular diffusion channels. Combined with the extended enzyme molecule residence time caused by the low flow rate, collagenase P can cross the hepatic sinusoidal barrier and evenly penetrate into the collagen fiber network inside the liver lobules. It can complete the precise release of deep hepatocytes under a low enzyme load, effectively avoiding the phenomenon of edge over-digestion and core under-digestion common in traditional processes. Attached Figure Description

[0018] Figure 1This is a flowchart of the efficient perfusion preparation process of primary rodent hepatocytes according to the present invention; Figure 2 This is a schematic diagram of the liver tissue hierarchical permeation enzymatic hydrolysis path and the principle of global uniform tissue dissociation in this invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] It should be noted that all directional and positional terms used in this invention, such as: up, down, left, right, front, back, vertical, horizontal, inner, outer, top, bottom, transverse, longitudinal, center, etc., are only used to explain the relative positional relationship and connection between components in a specific state (as shown in the accompanying drawings). They are only for the convenience of describing this invention and do not require that this invention be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention. In addition, the descriptions of "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0021] In the description of this invention, unless otherwise explicitly specified and limited, the terms installation, connection, and linking should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to the internal connection of two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood in conjunction with the specific circumstances.

[0022] In the description of this specification, references to the terms "an embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0023] A highly efficient process for preparing perfusion fluid for primary rodent hepatocytes includes the following steps: Step S101: Pre-perfusion fluid preheated to 37°C and with a pH of 7.4 is pumped into the isolated liver of a rodent. The pumping flow rate is set to the first flow rate and is maintained for 2 minutes. The pre-perfusion fluid contains 0.92 g / L ascorbic acid, 0.5 g / L HEPES, 0.19 g / L EDTA, and DPBS buffer. Ascorbic acid removes reactive oxygen free radicals in the interstitial spaces of the liver tissue under the mechanical shearing environment generated by the first flow rate, and works with EDTA to chelate intercellular calcium ions to loosen the hepatocyte connections. Step S102: Pump DPBS buffer at 37°C into the liver, maintaining the first flow rate for 1 minute, and replacing residual EDTA and metabolites in the liver tissue with DPBS buffer. Step S103: Preheated digestive fluid to 37°C is pumped into the liver for stratified dissociation. The pumping flow rate is set to the second flow rate and is maintained for 3 minutes. The digestive fluid contains 10 mg / L hyaluronidase, 200 mg / L collagenase P, and basal culture medium. The second flow rate is 0.5 times that of the first flow rate. Hyaluronidase degrades glycosaminoglycans in the liver tissue matrix to establish a penetration channel for collagenase P into the deep layers of the liver lobules. Step S104: Immerse the liver tissue after hierarchical dissociation in a stop solution at 4°C. The stop solution contains 5% fetal bovine serum and 1% penicillin / streptomycin mixture by volume.

[0024] Preferably, in step S101, the concentration of EDTA in the pre-perfusion fluid is 0.5 mmol / L.

[0025] Preferably, the concentration of ascorbic acid in the pre-perfusion fluid is from 0.1 mmol / L to 0.5 mmol / L.

[0026] Preferably, in step S101, the DPBS buffer contains NaCl at a concentration of 130 mmol / L to 145 mmol / L and [unclear - possibly a specific ingredient or concentration] at a concentration of 5.0 mmol / L to 6.0 mmol / L. And concentrations of 1.0 mmol / L to 1.5 mmol / L .

[0027] Preferably, in step S103, the digestive fluid is pre-treated with 0.22... Microporous membrane filtration for sterilization.

[0028] Preferably, in step S103, the basal culture medium is DMEM culture medium.

[0029] Preferably, the pH of the termination solution is 7.3 to 7.5.

[0030] Preferably, after step S104, the method further includes: step S105, peeling off the liver capsule with ophthalmic forceps and releasing hepatocytes into the termination solution with mechanical vibration; step S106, filtering the termination solution through a 100-mesh nylon filter, collecting the filtrate and centrifuging it at 4°C, with the centrifugation speed set to 50g to 100g and the centrifugation time to 2min to 5min.

[0031] Preferably, after centrifugation, the cell pellet is resuspended in a washing solution and centrifuged 2 to 3 times to ensure that the viability of the obtained rodent primary hepatocytes is not less than 95%.

[0032] Example 1: In the industrial-grade rodent ex vivo liver perfusion process for high-throughput drug toxicology evaluation, obtaining a large number of highly active primary hepatocytes faces severe constraints due to mechanical shear stress and drastic fluctuations in the biochemical microenvironment. Because the collagen fiber network in the liver tissue of rodents of a certain age is highly cross-linked and the lipid bilayer of the sinusoidal endothelial cell membrane is extremely fragile, the instantaneous physical shear force generated by the perfusion fluid pump during the routine blood washing and decalcification stages can easily induce an explosion of reactive oxygen free radicals in the liver tissue interstitial space. This results in a large number of hepatocytes undergoing oxidative stress and irreversible submicroscopic structural damage before entering the collagenase degradation matrix step. This increased membrane fragility during the decalcification loosening process weakens cell viability and leads to high instability of receptor function in the final batch.

[0033] To resolve the physicochemical contradiction between in vitro stress injury and efficient dissociation, a pre-perfusion fluid preheated to 37°C and pH 7.4 was pumped into the isolated liver of a rodent. The pumping flow rate was set to the first flow rate and maintained for 2 minutes. The pre-perfusion fluid contained 0.92 g / L ascorbic acid, 0.5 g / L HEPES, 0.19 g / L EDTA, and DPBS buffer. Ascorbic acid rapidly scavenged reactive oxygen species in the interstitial spaces of the liver tissue under the mechanical shearing environment generated by the first flow rate, constructing a redox buffer barrier to maintain the physicochemical stability of the phospholipid layer of the hepatocyte membrane. EDTA chelated intercellular calcium ions to loosen the hepatocyte connections. DPBS buffer at 37°C was then pumped into the liver, maintaining the first flow rate for 1 minute. The remaining EDTA and metabolites in the liver tissue were replaced by a digestion solution preheated to 37°C containing 10 mg / L hyaluronidase, 200 mg / L collagenase P, and basal culture medium for stratified dissociation. The pump flow rate was set to 0.5 times the first flow rate and maintained for 3 minutes. By reducing the flow rate, the residence time of enzyme molecules in the liver tissue interstitial space was increased. Hyaluronidase was used to degrade glycosaminoglycans in the liver tissue matrix to establish a permeation channel for collagenase P to diffuse into the deep layers of the liver lobules. This allowed collagenase P to cross the sinusoidal barrier and permeate evenly into the internal collagen fiber network. Global dissociation from the inside out was achieved under a lower enzyme load, thereby eliminating the spatial inhomogeneity caused by peripheral over-digestion and core under-digestion that was caused by solely relying on increasing the collagenase concentration.

[0034] During the preparation of the pre-perfusion solution, 0.5 g of HEPES and 0.19 g of EDTA were added sequentially to 800 mL of DPBS buffer. The magnetic stir bar was kept at 200 rpm to accelerate the uniform dispersion of the solute. 0.92 g of ascorbic acid powder was weighed and added to the mixture. The pH of the system was adjusted to 7.4 by titration with 1 mol / L sodium hydroxide solution. The remaining volume was made up to 1 L with DPBS buffer. The resulting solution was sterilized by passing it through a 0.22 μm polyethersulfone filter membrane. The finished solution was stored at 4°C to maintain the reduced state of ascorbic acid. After the cascade dissociation was completed... Subsequently, the isolated liver tissue was directly immersed in a stop solution containing 5% fetal bovine serum and 1% penicillin / streptomycin mixture at 4°C. The digestion reaction was interrupted by the low-temperature physical environment and the protease inhibitor in the serum. After subsequent mechanical shaking and centrifugation, the surface microvilli structure of the obtained rodent primary hepatocytes showed a highly regular distribution and no signs of vacuolation. The trypan blue rejection rate test showed that the cell viability was maintained at a level of not less than 95%. The overall process reduced the comprehensive preparation cost of high-activity primary cells and established the unit viable cell production efficiency that meets the requirements of large-scale drug screening.

[0035] Example 2: In the process of acquiring primary rodent hepatocytes for high-throughput toxicology evaluation, the mechanical shear noise caused by ambient temperature drift and the periodic pulsation of the peristaltic pump continuously disturbed the cell viability data. To quantify the impact of fluid control and biochemical environment configuration on cell dissociation efficiency, physical and biological data were acquired using an ex vivo organ isothermal perfusion platform with online flow rate monitoring and real-time dissolved oxygen detection. This platform was preset with a baseline flow rate pulsation of 20 dB signal-to-noise ratio to simulate an industrial fluid environment. For the key process parameter of digestive fluid inflow rate, the logic was set to achieve a balance between the residence time of enzyme molecules in the interstitial space and the fluid shear stress. When the liver tissue has a high vascular network density and a dense extracellular matrix, in order to avoid mechanical tearing of endothelial cells caused by high flow rate and to ensure the contact area of ​​enzymatic reaction, the digestive fluid inflow rate must tend to the downward range of the pre-perfusion flow rate. Based on this, the parameter judgment rule of digestive fluid inflow rate being 0.5 times the first flow rate was determined, and the mass transfer time of biochemical degradation was obtained by reducing the fluid kinetic energy input.

[0036] To quantify the physical and biochemical synergistic effects of the aforementioned process parameters, an experimental group using the complete perfusion fluid components and a set flow rate was established. Simultaneously, a first control group (hyaluronidase stripping), a second control group (ascorbic acid concentration reduced to 0.10 g / L), and a third control group (ascorbic acid concentration increased to 2.50 g / L) were also established. All groups were pumped with perfusion fluid into a perfusion platform with baseline flow rate pulsation. Monitoring data showed that in the second control group, the trypan blue rejection rate was only 72.4% due to reactive oxygen species stress induced by fluid shear. In the third control group, the cell viability dropped to 81.3% due to osmotic pressure imbalance caused by high ascorbic acid concentration leading to cell membrane rupture. In contrast, the experimental group achieved a viability of 95.6% at an ascorbic acid concentration of 0.92 g / L. Quantitative data demonstrate… 0.92 g / L maintained the balance between antioxidant and osmotic pressure and exhibited a nonlinear performance inflection point. The first control group lacked glycosaminoglycan degradation channels, resulting in impaired collagenase penetration and lower cell viability and dissociation uniformity in deep tissues compared to the experimental group. The experimental group, with the synergistic effect of 10 mg / L hyaluronidase and 200 mg / L collagenase P, combined with flow rate attenuation control, ensured that the enzymatic hydrolysate covered the liver lobule structure, inhibiting shear interference caused by baseline flow pulsation. Within the set temperature and flow rate physical boundaries, ascorbic acid and EDTA in the pre-perfusion fluid cooperated to continuously operate the hierarchical permeation enzymatic hydrolysis pathway. The coupling balance between biochemical distribution ratio and hydrodynamic parameters balanced in vitro stress damage and tissue dissociation efficiency, and stably output cell batches with a viability of 95.6%.

[0037] Example 3: This example combines Figures 1 to 2 This document describes a highly efficient process for preparing perfusion fluid for primary rodent hepatocytes. Figure 1As shown, step S101 corresponds to pumping in pre-perfusion fluid to remove free radicals and loosen the connection. The process proceeds to step S102, which corresponds to pumping in buffer solution to replace residual EDTA and metabolites. Then, the process proceeds to step S103, which corresponds to pumping in digestion fluid to degrade the matrix and build permeable channels. Finally, the process proceeds to step S104, which corresponds to immersing the dissociated liver tissue in the stop solution.

[0038] like Figure 2 As shown, the dissociation process includes preliminary dissociation on the left: the hyaluronidase action region, and core dissociation on the right: the collagenase P deep penetration region. In the preliminary dissociation region, the upper primitive is the degradation of tissue matrix glycosaminoglycans, which points downward to open molecular diffusion and penetration channels. The primitive that opens molecular diffusion and penetration channels connects laterally to the core dissociation region to uniformly penetrate across the liver sinusoidal barrier. This step then points downward to degrade the deep collagen fiber network of the liver lobules. From the above-mentioned core dissociation region, the process points downward to the final output stage, corresponding to the realization of global uniform dissociation of liver tissue to preserve cell receptor activity and viability.

[0039] Example 4: In the batch processing of isolated rodent livers with varying physiological differences, fixed process parameters caused fluctuations in cell yield. Addressing the challenges of unclear mass transfer mapping of components in the pre-perfusion fluid at the sinusoidal endothelial interface, the lack of a quantitatively based centrifugation purification procedure for cell separation after digestion termination, and the absence of a dynamic adjustment mechanism for the digestion flow rate based on the initial physical state of the liver, an integrated control logic based on dynamic speed regulation of initial portal vein resistance and multi-stage differential centrifugation was established. A perfusion catheter was connected to the portal vein of the isolated rodent liver, and 37°C DPBS buffer was pumped in while maintaining a baseline flow rate of 10 mL / min. A pressure sensor connected in series in the tubing acquired the initial portal vein resistance value. Based on the deviation ratio of the initial portal vein resistance value from the standard resistance baseline, the pumping rates of the pre-perfusion fluid and digestion solution were simultaneously adjusted. When the initial portal vein resistance value exceeded the standard resistance baseline, the first flow rate of the pre-perfusion fluid was set as shown in the formula: ,in, The first flow rate after adjustment. To calibrate the first flow velocity, This represents the vascular compliance damping coefficient. The value represents the difference between the initial portal vein resistance value and the standard resistance baseline. The rate-adjustment logic, combined with the correspondingly increased perfusion time, ensures that a unit volume of liver tissue receives a constant molar amount of ascorbic acid and EDTA during the pre-perfusion phase. Under a defined fluid kinetic energy, ascorbic acid covers the lipid bilayer of endothelial cells and neutralizes reactive oxygen free radicals. EDTA molecules penetrate into the intercellular space and chelate calcium ions in cadherin to maintain the osmotic pressure gradient within the liver lobules. The mechanical shear force generated by the macroscopic fluid flow acts on the microvascular network, triggering transient stress in the cell wall to generate oxygen free radicals. The ascorbic acid molecules, in a completely dissolved state, simultaneously cross the laminar boundary and adhere to the outer side of the cell membrane through the convective mass transfer mechanism. With its extremely high biochemical diffusion flux, it offsets the reaction delay between the macroscopic volumetric flow rate and the microscopic molecular collision, thereby achieving kinetic synergy across spatial scales.

[0040] The digestion solution used Williams' Medium E basal medium preheated to 37°C as the solvent. 10 mg of hyaluronidase powder and 200 mg of collagenase P were added to 1 L of Williams' Medium E basal medium, and the stirring intensity was controlled until the solute was completely eliminated. The calcium ion concentration in Williams' Medium E basal medium was maintained at 1.8 mmol / L to provide the catalytic cofactor required for collagenase P to degrade the collagen fiber network. This solvent environment, together with hyaluronidase, opened up deep tissue penetration pathways and controlled the metabolic acid load during digestion within the cell physiological tolerance threshold.

[0041] Digestive solutions containing hyaluronidase and collagenase P were used to perform stratified dissociation at an adjusted flow rate. The isolated liver tissue was transferred to a sedimentation dish containing a 4°C stop solution, which included 5% fetal bovine serum and a 1% mixture of penicillin and streptomycin. The dissociated cell suspension was filtered through a 100μm nylon mesh to intercept undigested connective tissue framework. The filtrate was transferred to centrifuge tubes and placed in a low-temperature centrifuge. The centrifuge chamber temperature was set to 4°C, and the rotor speed provided a relative centrifugal force of 50g for sedimentation acceleration. Centrifugation lasted for 3 minutes. The centrifugation procedure utilized the density difference between primary hepatocytes and non-parenchymal cells, driving the high-density primary hepatocytes through the stop solution medium to the bottom of the tube under a relative centrifugal force of 50g, while the low-density non-parenchymal cells and matrix debris were suspended in the supernatant. The supernatant was discarded, and the hepatocyte pellet was resuspended in 4°C DPBS buffer. The centrifugation process was repeated twice to replace residual proteases and tissue debris in the system.

[0042] Dynamic flow rate compensation based on initial portal vein resistance value, combined with multi-stage low-speed differential centrifugation procedures, avoids mass transfer inhomogeneity and secondary shear damage during the dissociation process of livers with different physiological backgrounds. Quantitative tests show that the non-parenchymal cell contamination rate in the isolated primary hepatocyte population is no higher than 2%, and the cell membrane structure remains intact, establishing a physical boundary for the extraction of highly active hepatocytes with process reproducibility.

[0043] Example 5: When facing the initial conditions of liver dissection in rodents of different ages and strains, 4°C calcium-free basal buffer was pumped into the isolated liver connected to the perfusion catheter. The pumping rate was linearly increased from 2 mL / min to 10 mL / min. Portal vein pressure data were collected at the corresponding flow rate nodes. The standard resistance baseline was obtained by fitting the slope of the flow rate and pressure data. The coordinates of the yield point on the pressure curve that deviated from the linear interval were extracted. The vascular compliance damping coefficient was calculated based on the flow rate decrease corresponding to the yield point. ,in The yield point coordinates are dimensionless vascular compliance damping coefficients. The system calculates the transient pressure change slope corresponding to each flow velocity node and compares it with the standard resistance baseline. When the transient pressure change slope drops below 80% of the standard resistance baseline for three consecutive sampling cycles, the system locks the current flow velocity node as the yield point coordinate. The physical time span of a single pressure sampling cycle within the calculation module is preset to 500ms. This time limit establishes a high-frequency data interception window to avoid peristaltic pump mechanical output pulses. The system extracts the corresponding flow velocity attenuation and portal vein pressure rise at the node. The flow velocity attenuation divided by the portal vein pressure rise yields the vascular compliance damping coefficient. The system characterizes the limit of microvascular physical compensatory expansion caused by unit pressure changes. The platform control module outputs a linear temperature rise command to the heating component in the input tubing. The heating component drives the temperature of the calcium-free basal buffer solution to rise from 4°C to 37°C at a constant rate of 5°C / min. The system maintains a constant temperature of 37°C and continues in situ circulation perfusion for 3 minutes to limit the superimposed fluctuations in temperature jumps and endothelial cell deformation stress. The platform control module stores the standard resistance baseline and the vascular compliance damping coefficient. Used as the baseline parameter for the current batch being processed.

[0044] The platform control module receives the standard resistance baseline and the vascular compliance damping coefficient. As a flow rate regulation constraint, the initial portal vein resistance value during the actual pumping phase is monitored, and the difference between the initial portal vein resistance value and the standard resistance baseline is calculated. ,in This refers to the portal vein pressure difference; the platform control module is based on... With vascular compliance damping coefficient A flow rate compensation command is generated to drive the peristaltic pump servo motor to reduce its speed and decrease the output flow rate of the pipeline. The flow rate compensation command constrains the physical shear stress generated by the pumping of perfusion fluid under different physiological conditions, and maintains the consistency of the fluid dynamics inside the isolated liver.

[0045] Example 6: When the system faces the initial condition of liver dissociation in rodents of different ages and strains, 4°C calcium-free basal buffer is pumped into the isolated liver connected to the perfusion catheter. The pumping rate is linearly increased from 2 mL / min to 10 mL / min. Portal vein pressure data are collected at the corresponding flow rate nodes. The standard resistance baseline is obtained by fitting the slope of the flow rate and pressure data. The coordinates of the yield point on the pressure curve that deviates from the linear interval are extracted. The vascular compliance damping coefficient is calculated based on the flow rate decrease corresponding to the yield point. ,in The dimensionless vascular compliance damping coefficient; the platform control module stores the standard resistance baseline and the vascular compliance damping coefficient. Used as the baseline parameter for the current batch being processed.

[0046] The platform control module receives the standard resistance baseline and the vascular compliance damping coefficient. As a flow rate regulation constraint, the initial portal vein resistance value during the pumping phase is monitored, and the difference is calculated by subtracting the standard resistance baseline from the initial portal vein resistance value. ,in This refers to the portal vein pressure difference; the platform control module is based on... With vascular compliance damping coefficient A flow rate compensation command is generated to drive the peristaltic pump servo motor to adjust its speed to reduce the output flow rate of the pipeline. The flow rate compensation command constrains the physical shear stress generated by the pumping of perfusion fluid under different physiological backgrounds, maintaining the consistency of the fluid dynamics inside the isolated liver.

[0047] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A highly efficient process for preparing perfusion fluid for primary rodent hepatocytes, characterized in that, Includes the following steps: Step S101: Pre-perfusion fluid preheated to 37°C and pH 7.4 is pumped into the isolated liver of a rodent. The pumping flow rate is set to the first flow rate and is maintained for 2 minutes. The pre-perfusion fluid contains 0.92 g / L ascorbic acid, 0.5 g / L HEPES, 0.19 g / L EDTA, and DPBS buffer. Ascorbic acid removes reactive oxygen free radicals in the interstitial spaces of the liver tissue under the mechanical shearing environment generated by the first flow rate, and works with EDTA to chelate calcium ions between cells to loosen the connections between liver cells. Step S102: Pump DPBS buffer at 37°C into the liver, maintaining the first flow rate for 1 minute, and replacing residual EDTA and metabolites in the liver tissue with DPBS buffer. Step S103: Preheated digestive fluid to 37°C is pumped into the liver for stratified dissociation. The pumping flow rate is set to the second flow rate and is maintained for 3 minutes. The digestive fluid contains 10 mg / L hyaluronidase, 200 mg / L collagenase P, and basal culture medium. The second flow rate is 0.5 times that of the first flow rate. Hyaluronidase degrades glycosaminoglycans in the liver tissue matrix to establish a penetration channel for collagenase P into the deep layers of the liver lobules. Step S104: Immerse the liver tissue after hierarchical dissociation in a stop solution at 4°C. The stop solution contains 5% fetal bovine serum and 1% penicillin / streptomycin mixture by volume.

2. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, In step S101, the concentration of EDTA in the pre-perfusion fluid is 0.5 mmol / L.

3. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, The concentration of ascorbic acid in the pre-perfusion fluid was 0.1 mmol / L to 0.5 mmol / L.

4. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, In step S101, the DPBS buffer contains NaCl at a concentration of 130 mmol / L to 145 mmol / L and [unclear - possibly a specific ingredient or concentration] at a concentration of 5.0 mmol / L to 6.0 mmol / L. And concentrations of 1.0 mmol / L to 1.5 mmol / L .

5. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, In step S103, the digestive fluid is pre-pumped via a 0.22... Microporous membrane filtration for sterilization.

6. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, In step S103, the basal culture medium is DMEM medium.

7. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, The pH of the stop solution is 7.3 to 7.

5.

8. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 1, characterized in that, Step S104 is followed by: Step S105, peeling the liver capsule with ophthalmic forceps and releasing hepatocytes into the termination solution with mechanical vibration; Step S106, filtering the termination solution through a 100-mesh nylon filter, collecting the filtrate and centrifuging it at 4°C, with the centrifugation speed set to 50g to 100g and the centrifugation time to 2min to 5min.

9. The process for preparing perfusion fluid for efficient preparation of primary rodent hepatocytes according to claim 8, characterized in that, After centrifugation, the cell pellet was resuspended in washing solution and centrifuged 2 to 3 times to ensure that the viability of the obtained rodent primary hepatocytes was not less than 95%.

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