CN114045255A

CN114045255A - Method for separating and culturing renal tubular epithelial cells

Info

Publication number: CN114045255A
Application number: CN202111397633.7A
Authority: CN
Inventors: 樊琛语; 刘小燕; 邱江; 王恒; 郑立新
Original assignee: Guangzhou Huayue Nephrology Regenerative Medicine Technology Co ltd
Current assignee: Guangzhou Huayue Nephrology Regenerative Medicine Technology Co ltd
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-02-15
Anticipated expiration: 2041-11-23
Also published as: CN114045255B

Abstract

The invention discloses a method for separating and culturing renal tubular epithelial cells, which comprises the following steps: s1: mechanical tissue separation: collecting kidney tissue sample, separating cortex part, and cutting; s2: enzymolysis: digesting and centrifuging by using mixed enzyme; s3: cell culture: the cell pellet was resuspended in complete medium and inoculated into a culture flask. Wherein the mechanical separation is simple and the mechanical damage is reduced to the maximum extent. The mild enzymolysis achieves the best digestion efficiency, the obtained cells have high activity, the activity of the obtained cells can reach more than 50 percent, and the cell number at least reaches 1 multiplied by 10⁶The seed cells per gram have high proliferation and high initial inoculation density, so that the proliferation environmental pressure of the cells is low, and the cell proliferation speed is at least doubled compared with that reported in the literature due to the addition of a nutrient-rich and simple nutrient medium without matrix layer plating; e.g. 1 x 10⁶Cells were seeded in T75 flasks and complete confluency was achieved for 72 h.

Description

Method for separating and culturing renal tubular epithelial cells

Technical Field

The invention belongs to the technical field of cell extraction, and particularly relates to a method for separating and culturing renal tubular epithelial cells.

Background

The kidney is a complex organ, mainly composed of glomeruli, tubules, mesangial cells, endothelial cells and podocytes. The tissues and cells are separated and purified from organs, and then the primary cells are cultured in vitro, so that the state of the cells in vivo can be truly reflected, and the expression of functional proteins and markers is complete, which is an indispensable part in basic research. In addition, with the rise of various kidney disease-related therapeutic drugs, the development of in vitro cultured cell systems as the most straightforward and simple drug safety detection platform should be considered. Several protocols exist for isolating these cells, the most isolated of which are the proximal tubular epithelial cells. They play an important role in water balance, acid-base control, reabsorption of chemical molecules, secretion of foreign substances and endogenous metabolites, etc.

In the past, renal toxicity research and safety evaluation related to medicines are carried out on animals, and related in vitro research results are difficult to apply directly to human bodies. Human-related experiments are not only cost-limited, but also more ethical. One approach to solving these problems is 2D kidney cell culture. Specific cell types such as tissues such as glomeruli and renal tubules, mesangial cells, podocytes and the like can be separated according to different test objects, drug targets and the like, and the cell types can be researched in a targeted manner. Isolation of primary cells from kidney tissue remains the gold standard for current in vitro test models, given the genomic and epigenetic changes that cell immortalization and long-term cell passaging techniques may result in.

The invention application patent CN109402041A discloses a method for separating and culturing human renal tubular epithelial cells, which comprises the following steps: (a) washing the tissue specimen with double-anti precooling PBS; (b) cutting and grinding tissues, and collecting ground tissue blocks; (c) digesting with mixed enzyme (trypsin, collagenase IV and collagenase I), filtering with a screen, collecting filtrate, and centrifuging; (d) separating Percoll; (e) suspending the cell sediment by a complete culture medium, and inoculating the cell sediment into a culture bottle; (f) after several days, the mixed complete culture medium is replaced for continuous culture.

The enzymatic digestion is a method commonly used for isolating single cells from tissues, and commonly used enzymes include trypsin, collagenase, hyaluronidase, and neutral protease (dispase). Because each tissue has different structures and compositions, and the same tissue has different species and genera, different enzymes have different digestion efficiencies for different tissues. Enzymes with strong digestive ability such as pancreatin easily damage cells (uneven digestion). During the process of pancreatin digestion, part of cells are not digested yet, and a small part of cells are over-digested, but when the pancreatin concentration does not reach the effective concentration, the tissue cells can not be effectively digested. The time for pancreatin to digest tissue cells from complete digestion to excessive digestion is short, it is difficult to control the digestion time, the yield of incomplete digestion is low, excessive digestion causes cell injury, the cell viability is reduced, and cells in the same state cannot be stably obtained.

Disclosure of Invention

The invention aims to shorten the time of enzyme digestion of the renal cortex tissue, prolong the time from complete digestion to just over digestion as far as possible, improve the controllability of cell separation of the digested renal cortex tissue and avoid over digestion. A simple and efficient method for separating and culturing renal tubular epithelial cells comprises two digestion steps. The method is suitable for separating and culturing renal tubular epithelial cells from renal cortex tissues of human, monkey and pig. The first preliminary digestion not only shortens the time for the second digestion, but also extends the controlled time (from complete digestion to just over digestion). In addition, the method has mild condition, little damage to cells, large number of extracted cells, high activity, high proliferation speed of the obtained renal tubular epithelial cells and high passable times.

The invention aims to provide a method for separating and culturing renal tubular epithelial cells.

The purpose of the invention is realized by the following technical scheme:

a renal tubular epithelial cell separation and culture method comprises the following steps:

s1: mechanical tissue separation: segmenting renal cortical tissue into tissue fragments;

s2: enzymolysis: digesting the tissue fragment by using a first digestive enzyme, centrifuging to remove a supernatant, and digesting by using a second digestive enzyme to obtain a cell;

s3: cell culture: inoculating the obtained cells into a culture medium for culture;

the first digestive enzyme comprises dispase and collagenase;

the second digestive enzyme is collagenase.

In some embodiments of the invention, the collagenase of the first and second digestive enzymes is collagenase type II or collagenase type IV.

In some embodiments of the invention, the concentration of the dispersing enzyme in the first digestive enzyme is 0.75-1.5 mg/mL, and the concentration of the collagenase is 1-2.5 mg/mL.

In some embodiments of the invention, the concentration of the second digestive enzyme is 1-2.5 mg/mL.

In some embodiments of the invention, the ratio of the dispase and the collagenase of the first digestive enzyme is 1: 0.5-2 by volume.

In some embodiments of the present invention, the time for the first digestive enzyme to digest the tissue is 45-75 min, and the time for the second digestive enzyme to digest the tissue is 40-60 min.

In some embodiments of the present invention, in step S2, 2-3 mL of the first digestive enzyme is added per gram of the tissue fragment, and 0.5-0.1 mL of the second digestive enzyme is added per gram of the tissue fragment.

In some embodiments of the present invention, the tissue fragment of step S1 is 1-3 mm³。

In some embodiments of the present invention, the cell seeding density in step S3 is 0.5-1.5 × 10⁶Per 75m²。

In some embodiments of the invention, the centrifugation conditions in step S2 are: centrifuging at 800-1200 rpm for 4-10 min.

In some embodiments of the invention, the centrifugation conditions in step S2 are: centrifuge at 1000rpm for 5 min.

In some embodiments of the present invention, for better preservation of cell activity, the centrifugation preferably occurs in a low temperature environment, preferably 3-10 ℃, more preferably 4 ℃.

The invention has the beneficial effects that:

(1) the digestion time is relatively short, the time from complete digestion to excessive digestion of tissue cells is long, the digestion time is convenient to control, and excessive digestion is not easy to cause;

(2) the method of mechanical separation and enzymolysis separation is mild, and the obtained cells have high survival rate (more than 50 percent) and good state (high proliferation);

(3) the obtained renal tubular epithelial cells do not need matrix layer plating culture, and have high proliferation speed (1 × 10)⁶The primary generation cells are inoculated in a T25 bottle, complete confluence can be achieved within 72 hours), and the passability times are high.

(4) The operation is simple, and equipment such as a stainless steel mesh screen, grinding equipment and percoll gradient centrifugal separation is not required to be matched.

Drawings

FIG. 1 shows that P0 cells reached 100% confluence within 72 h.

FIG. 2 shows the confluent state of tubular epithelial cells isolated and cultured in example 1, wherein P1 is the first generation of cells, P4 is the fourth generation of cells and P6 is the sixth generation of cells.

FIG. 3 is the state of P6 generation renal tubular epithelial cells of example 1.

FIG. 4 shows the cells obtained in example 2 by digesting with dispase and collagenase type II for 60min and then collagenase type IV for 45 min. Wherein FIG. 4A is a cell growth status; FIG. 4B shows cell viability.

FIG. 5 shows the confluent state of the P0 generation tubular epithelial cells in example 2.

FIG. 6 shows the cells obtained in example 3 by digesting with dispase and collagenase type IV for 70min and then collagenase type II for 45 min. Wherein FIG. 6A is a cell growth status; FIG. 6B shows cell viability.

FIG. 7 shows the cells obtained in comparative example 1 after 10min of trypsinization.

FIG. 8 shows the cells obtained by collagenase II digestion for 4h in comparative example 2.

FIG. 9 shows the cell viability of the papain digestion of comparative example 4 for 30 min.

FIG. 10 shows cells digested with collagenase type II and dispase of comparative example 5 for 3 h.

FIG. 11 shows cells obtained by digesting 105min with collagenase type II and dispase of comparative example 6.

FIG. 12 shows the cells obtained by digesting 105min with collagenase type II and dispase of comparative example 6, and centrifuging to extract 1 x 10⁶The cells were inoculated in a culture medium, and the P0 generation renal tubular epithelial cells were in a confluent state.

FIG. 13 shows cells obtained by digesting 105min with collagenase type IV and dispase of comparative example 7.

FIG. 14 shows the cells obtained by digesting 105min with collagenase type IV and dispase of comparative example 7, and centrifuging to extract 1X 10⁶The cells were inoculated in a culture medium, and the P0 generation renal tubular epithelial cells were in a confluent state.

FIG. 15 shows cells from 30min digestion with collagenase type II and papain according to comparative example 8.

FIG. 16 shows cells obtained by digesting 30min with dispase and papain in comparative example 9.

FIG. 17 shows cells obtained by digesting the dispase and collagenase type II of comparative example 10 for 1 hour and then digesting them with pancreatin for 5 min.

FIG. 18 shows cells obtained by digesting the dispase and collagenase type II of comparative example 11 for 1 hour and then digesting them with pancreatin for 10 min. Wherein FIG. 18A is the cell growth state; fig. 18B is cell viability.

FIG. 19 shows cells obtained by digesting the dispase and collagenase type II of comparative example 12 for 3 hours and then with papain for 30 min.

FIG. 20 shows cells obtained from collagenase type II and dispase of comparative example 13 digested for 4 hours and then digested with papain for 30 min.

FIG. 21 shows cells obtained by digesting 30min with collagenase type II, papain and dispase of comparative example 14.

FIG. 22 shows the cell viability of cells obtained from comparative example 18 after collagenase IV digestion for 1h, followed by dispase and collagen IV digestion for 30 min.

FIG. 23 shows the state of cells obtained by sieving in comparative example 23 and gradient density centrifugation of Percoll cell separation medium, in which FIG. 23A shows the state after sieving, FIG. 23B shows the state after viability detection after sieving, and FIG. 23C shows the state after gradient density centrifugation.

FIG. 24 shows the state of P0 cells extracted by sieving in comparative example 23 and gradient density centrifugation of Percoll cell separation medium.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1 isolation and culture of renal Primary tubular epithelial cells

(1) Pretreatment of renal cortex: the washed human kidney tissue is put into a stainless steel tray filled with PBS at 4 ℃, envelope, fat and the like are removed by tissue scissors and tweezers, the kidney is cut from the middle by a surgical knife, the cortex is cut along the color boundary of the cortex and the medulla, the cortex is completely separated, the separated cortex is put into a 50mL centrifuge tube filled with PBS at 4 ℃, the washing is thorough, the supernatant is sucked by a pipette, and the operation is repeated for a plurality of times until no bloodstain exists in the solution (if the tissue is the cortex, the cortex is only cut into pieces). After the remaining tissue blocks are weighed and recorded, the remaining renal cortex is cut into 1-3 mm by using surgical scissors³The smallest pieces are cut as far as possible. Soaking in a culture dish containing 4 deg.C PBS, placing an ice bag at the bottom of the culture dish, placing the separated cortex into a centrifuge tube containing 4 deg.C PBS, thoroughly cleaning, removing the supernatant with a pipette, and repeating for several times until there is no blood stain in the solution.

(2) Enzymolysis and digestion:

placing digestive enzyme into a centrifuge tube filled with the kidney cortex tissue fragment for digestion: mixing dispase (1mg/mL) and type II collagenase (2mg/mL) at a volume ratio of 1:1, adding 2mL of the above enzyme mixture to 1g of the crushed pieces of renal cortex tissue, digesting the tissue for 50min, centrifuging to remove the supernatant, adding 0.75mL of type IV collagenase (2mg/mL), and further digesting for 45 min. The centrifugation conditions are as follows: centrifuge at 1000rpm for 5min at 4 ℃.

(3) Renal tubular epithelial cell plating and culture protocol:

in vitro culture of 2D pure tubular cells did not require plating of extracellular matrix components. The medium composition scheme is as follows: DMEM-F12+ 10% fetal bovine serum + 1% penicillin/streptomycin.

After the enzymolysis is finished, the supernatant is cleared of collagenase by centrifugation procedure. The cells were then resuspended in the above medium, filtered through a 100 μm cell sieve, the undersize cell pellet removed and cell viability measured (trypan blue), pellet density 1 x 10 in T75 flasks⁶Left and right. The culture flask was not moved for 2 days, and the cells were allowed to adapt to the in vitro culture state and gradually adhered to the flask wall.

As a result: cells reached 100% confluence within 72h (FIG. 1), during which time the medium was changed every 2 days (DMEM-F12+ 10% fetal bovine serum + 1% penicillin/streptomycin) and passaged. The dilution ratio of P1 to P2 is 1:3, and the growth can be completed in 3 days. The generation P3-P4 takes about 4-5 days to grow. After the P5 generation, the cell line enters the aging stage, the proliferation speed is reduced, and the cell line enters the aging stage and can grow up after 7 days, and the result is shown in figure 2. The highest P7(D9) can be reached, and the cells still maintain better morphology and growth rate after passage 6 (FIG. 3), and grow full in 15 days.

The cell obtained by the scheme has high cell activity, the separation method maximally reduces mechanical damage and mild enzymolysis, the survival rate of the obtained cell can reach 55 +/-5%, and the cell number extracted from the tissue at least reaches 1 multiplied by 10⁶(renal cortex tissue). The high initial inoculation density ensures that the proliferation environment pressure of the cells is less, and the proliferation speed of the renal tubular epithelial cells is higher without matrix layer plating by adding a simple nutrient medium which is rich in nutrition. E.g. 1 x 10⁶Cells were seeded in T75 flasks and complete confluency was achieved within 72 hours.

Example 2

The procedure for the isolation and culture of renal primary tubular epithelial cells in example 2 was the same as in example 1 except for the enzymatic digestion procedure.

And (3) enzymolysis and digestion operation: digesting the kidney cortex tissue fragment for 60min by adopting dispase (1mg/mL) and collagenase type II (2mg/mL) in the first digestion, centrifuging to remove the supernatant, and adding collagenase type IV (1mg/mL) in the second digestion for digesting for 45 min; the procedure was the same as in example 1 except that no DNA removal step was included.

As a result: the tissue fragments are digested as shown in figure 4, single cells can be effectively and completely separated, and the number of the cells is large. The obtained renal tubular epithelial cells have high survival rate on complete culture medium, cell activity can reach 60%, and the extracted cell number at least reaches 3 × 10⁶/g；1×10⁶The cells were seeded in T75 bottles and complete confluence could be achieved 2-3 days (FIG. 5, the texture was more uniform, less large cell clumps appeared), and the cells maintained good morphology and growth rate after passage 6. Experiments prove that the separation effect is still good when IV collagenase is added for digestion for 40min to 60min in the second digestion, the survival rate of the obtained renal tubular epithelial cells on a complete culture medium is kept between 55 percent and 65 percent, the digestion can be carried out for a controllable time of about 20 minutes, and the renal tubular epithelial cells can be conveniently and stably produced.

Example 3

The procedure for isolation and culture of renal primary tubular epithelial cells in example 3 was the same as in example 1 except for the enzymatic digestion procedure.

And (3) enzymolysis and digestion operation: digesting for 70min by using a mixed solution of dispase (1mg/mL) and collagenase type IV (1mg/mL) in the first digestion, centrifuging to remove a supernatant, and adding collagenase type II (2mg/mL) in the second digestion for digesting for 45 min; the procedure was the same as in example 1 except that no DNA removal step was included.

As a result: as a result, as shown in FIG. 6, it can be seen that the single cells can be efficiently and completely isolated and that the number of cells is large. The obtained cells have high survival rate on complete culture medium, the cell activity can reach 57%, and the cell number at least reaches 2 × 10⁶Per gram; 1X 10⁶The cells are inoculated in a T75 bottle, complete confluence can be achieved within 72 hours, and the cells still maintain good shape and growth rate after passage 6. Experiments prove that the separation effect is still good when II type collagenase is added for digestion for 40min to 60min in the second digestion, the survival rate of the obtained renal tubular epithelial cells is kept between 50 percent and 60 percent, the digestion can be carried out for a controllable time in about 20 minutes, and the renal tubular epithelial cells can be conveniently and stably produced.

Example 4 digestive enzyme optimization

The experiment of the embodiment aims to improve the efficiency of efficiently digesting the renal cortex tissue, obtain longer controllable digestion time and improve the yield and the cell activity. Isolation of renal primary tubular epithelial cells the method of culture was essentially the same as that of example 1, except for the conditions of enzymatic digestion.

Comparative example 1: the enzymolysis conditions are as follows: the renal cortex tissue fragment was digested with 0.25% pancreatin for 10min, and the procedure was otherwise the same as in example 1.

As a result: as shown in FIG. 7, it can be seen that the enzymatic action is too strong and the digestive juice is viscous. After groping, the cell digestion is completed and the cell enters the over-digestion stage immediately before and after 0.25 percent of pancreatin is digested for 10 min. Digestion can be controlled in a short time, which is not favorable for experimenters to control the time point of digestion, and results in unstable cell yield and activity.

Comparative example 2: the enzymolysis conditions are as follows: collagenase type II (2mg/mL) digested the renal cortex tissue fragment for 4 h. The other operations were the same as in example 1.

As a result: as in fig. 8, the long-term digestion still did not completely digest and separate the tissue into single cells.

Comparative example 3: the enzymolysis conditions are as follows: dispase (1mg/mL) digested fragments of renal cortical tissue for 4 h. The other operations were the same as in example 1.

As a result: long term digestion still does not completely digest and separate the tissue into individual cells.

Comparative example 4: the enzymolysis conditions are as follows: papain (2mg/mL) digested fragments of the renal cortex tissue for 30min, and the procedure was otherwise the same as in example 1.

As a result: cells were completely digested before and after 30 minutes, but cell viability was determined to be 15% (fig. 9); 1X 10⁶The cells were seeded in T75 flasks and complete confluence was achieved after 4-5 days. In addition, the time from complete digestion to excessive digestion is short, only 2-3 minutes is found, so that the time point for controlling digestion by experimenters is not facilitated, and the yield and activity among cells of each batch are not high.

Comparative example 5: the enzymolysis conditions are as follows: collagenase type II (2mg/mL) and dispase (1m/mL) were mixed together in a volume of 1:1 to digest the renal cortex tissue fragment for 3 h. The other operations were the same as in example 1.

As a result: referring to fig. 10, there were still a large mass of tissue in the suspension, too many cells were not seen under the microscope, and the digestion was incomplete.

Comparative example 6: the enzymolysis conditions are as follows: collagenase type II (2mg/mL) and dispase (1mg/mL) were mixed together in a volume of 1:1 to digest the kidney cortex tissue fragment for 105min, and the other operations were performed as in example 1.

As a result: as in fig. 11, digestion was incomplete. There are still a large number of cell clumps, not all of which become unicellular. The cells of the separated seed plates were grown to 100% confluence as shown in FIG. 12, and the P0 generation had many impurities and had uneven texture and size.

Comparative example 7: the enzymolysis conditions are as follows: the dispase (1mg/mL) and collagenase type IV (1mg/mL) were mixed at a volume of 1:1 to digest the kidney cortex tissue fragment for 105min and other procedures were performed as in example 1.

As a result: as in fig. 13, the digestion is not complete, there is a cell mass, not all of which become unicellular, and the cell mass is not clearly pasty and the cell is suspected of being partially ablated. The state of the separated seed plates when the cells were grown to 100% confluence is shown in FIG. 14, and the P0 generation still had cell masses and uneven texture and size.

Comparative example 8: the enzymolysis conditions are as follows: collagenase type II (1mg/mL) and papain (0.1mg/mL) were mixed at a volume ratio of 1:1 to digest the renal cortex tissue fragment for 30min, and the other operations were the same as in example 1.

As a result: the cells were not completely digested, see FIG. 15.

Comparative example 9: the enzymolysis conditions are as follows: dispase (1mg/mL) and papain (0.1mg/mL) were digested for 30 min. The other operations were the same as in example 1.

As a result: as can be seen in fig. 16, the digestion was incomplete and there were still many cell clumps.

Comparative example 10: the enzymolysis conditions are as follows: the first digestion is carried out by mixing and digesting the broken pieces of the renal cortex tissue for 1h by adopting dispase (1mg/mL) and type II collagenase (2mg/mL) according to the volume ratio of 1:1, centrifuging, discarding supernatant, adding 0.25% pancreatin, and carrying out second digestion for 5 min. The other operations were the same as in example 1.

As a result: referring to fig. 17, it can be seen that digestion is incomplete.

Comparative example 11: the enzymolysis conditions are as follows: the first digestion is carried out by digesting the kidney cortex tissue fragment for 1h by using a mixture of digestive enzyme (1mg/mL) and type II collagenase (2mg/mL), centrifuging, discarding the supernatant, and carrying out the second digestion for 10min by using 0.25% pancreatin. The other operations were the same as in example 1.

As a result: as in fig. 18, complete digestion can be seen under the mirror, but the total number of cells extracted was not high, with a survival rate of 60%. When 1X 10⁶The primary cells (P0) were seeded in T75 flasks and required 3-4 days to reach complete confluence. According to a plurality of comparative example data, the time for digesting the renal cortex by using the pancreatin is difficult to control, and the time from complete digestion to excessive digestion is too short, so that the cells can be damaged if the time is not well controlled.

Comparative example 12: the enzymolysis conditions are as follows: the first digestion is carried out by mixing collagenase II digestion (2mg/mL) and dispase (1mg/mL) according to the volume ratio of 1:1, after the kidney cortex tissue fragments are digested for 3 hours by magnetic stirring, the supernatant is centrifuged and discarded, and papain (0.1mg/mL) is added in the second digestion for further digestion for 30 minutes. The other operations were the same as in example 1.

As a result, referring to FIG. 19, it can be seen that the digestion time was long, the separation into single cells was not complete, and cell clusters were still visible.

Comparative example 13: the enzymolysis conditions are as follows: after the first digestion with collagenase type II (2mg/mL) and dispase (1mg/mL) in a volume of 1:1 mixed digestion (magnetic stirring) for 4h, the supernatant was discarded by centrifugation, and the second digestion with papain (0.1mg/mL) was continued for 30 min. The other operations were the same as in example 1.

As a result: referring to fig. 20, it can be seen that the cell membrane is ablated and dead cells are increased.

Comparative example 14: the enzymolysis conditions are as follows: separately, the fragment of the renal cortex tissue was digested with dispase (1mg/mL), collagenase type II (2mg/mL) and papain (0.1mg/mL) in a volume ratio of 1:1:1 for 30min, and the other operations were the same as in example 1.

As a result: referring to fig. 21, it can be seen that the digestion is not uniform, a portion of the tissue has not yet been digested, the digestive juice becomes viscous, another portion has been over-digested, and cell membrane ablation has occurred.

Comparative example 15: the enzymolysis conditions are as follows: the first digestion was performed with collagenase type II (2mg/mL) and dispase (1mg/mL), the kidney cortex tissue fragment was digested by mixing 1:1 in volume for 1h, the supernatant was discarded by centrifugation, and the second digestion was performed with dispase (1mg/mL) for 45 min. The other operations were the same as in example 1.

As a result: can be completely digested to obtain cells. However, through experiment, after the secondary digestion of the dispase, the optimum time is 45-50 min, only 5min is controllable, and after 50min, the digestion is excessive.

Comparative example 16: the enzymolysis conditions are as follows: the first digestion digested the kidney cortex tissue fragment with collagenase type II (2mg/mL) for 1h, centrifuged to discard the supernatant, and the second digestion digested with collagenase type II (2mg/mL) for 3 h.

As a result: the digestion is incomplete, and a large number of cell clusters still exist.

Comparative example 17: the enzymolysis conditions are as follows: the first digestion digested the kidney cortex tissue fragment with collagenase type II (2mg/mL) for 1h, centrifuged to discard the supernatant, and the second digestion digested with dispase (1mg/mL) for 2 h.

Comparative example 18: the enzymolysis conditions are as follows: the first digestion uses collagenase type IV (2mg/mL) to digest the kidney cortex tissue fragment for 1h, and the second digestion uses dispase (1mg/mL) and collagenase type IV to digest the kidney cortex tissue fragment for 30min according to the volume ratio of 1: 1.

As a result: can be completely digested to obtain cells. However, experiments prove that the secondary digestion of the dispase is suitable for 30-35 min, only controllable time of about 5min is needed, after 35min, the dispase is over digested, and the cell activity is low, about 33% (fig. 22).

Comparative example 19: the enzymolysis conditions are as follows: the first digestion is carried out by mixing and digesting the crushed pieces of the renal cortex tissue for 1h by adopting dispase (1mg/mL) and collagenase type II (2mg/mL) according to the volume ratio of 1:1, and directly adding collagenase type IV (2mg/mL) without centrifugation for further digestion for 2 h. The other operations were the same as in example 1.

As a result: the digestion is incomplete.

Example 5 enzyme time optimization

This example experiment was optimized for the first time for dispase and collagenase. The digestion time of the enzyme was adjusted and the isolation of primary tubular epithelial cells from kidney was essentially the same as the culture method and example 2.

Comparative example 20: the enzymolysis conditions are as follows: digesting the kidney cortex tissue fragment for 30min by adopting dispase (1mg/mL) and collagenase type II (2mg/mL) in the first digestion, centrifuging to remove the supernatant, and adding collagenase type IV (1mg/mL) in the second digestion for digesting for 120 min; no DNA removal step was included and the other operations were as in example 2.

As a result: the digestion is incomplete.

Example 6 optimization of enzyme concentration

The purpose of this example is to optimize the concentration conditions of the enzymes, and the isolation and culture method of renal primary tubular epithelial cells is basically the same as that of example 2, except that the type and concentration of the enzymes are changed.

Comparative example 21: the enzymolysis conditions are as follows: the first digestion was performed by digesting a fragment of the renal cortex tissue with collagenase type II (2mg/mL) and dispase (0.5mg/mL) in a volume ratio of 1:1 for 1h, centrifuging to remove the supernatant, and the second digestion was performed with collagenase type II (2mg/mL) for 90 min.

As a result: cells were completely digested for 90 minutes, but cell viability was determined to be 25%; 1X 10⁶The cells were seeded in T25 flasks and complete confluency was achieved for 4-5 days. In addition, the time from complete digestion to excessive digestion is found to be 85-110min, but the survival rate of cells is poor and is between 20-30%.

Comparative example 22: the enzymolysis conditions are as follows: the first digestion was performed by digesting the crushed pieces of the renal cortex tissue with collagenase type IV (2mg/mL) and dispase (2mg/mL) in a volume of 1:1 for 1h, centrifuging to remove the supernatant, and the second digestion was performed with collagenase type IV (2mg/mL) for 30 min.

As a result: cells were completely digested for 90 minutes, but cell viability was determined to be 40%; 1X 10⁶The cells were seeded in T25 flasks and complete confluency was achieved for 4-5 days. In addition, the time from complete digestion to excessive digestion is found to be 30-40min, and the survival rate of cells is poor and is 35-45%.

Example 7 optimization of mechanical separation conditions

In this example, different isolation methods were performed to isolate the renal cortex into single cells, and the cell status of the cells obtained under different isolation methods was examined:

comparative example 23: the separation conditions were: sieving + gradient density centrifugation of Percoll cell separation: the kidney cortex tissue is firstly passed through a stainless steel sieve (40 meshes), the kidney tissue on the sieve is slightly rotated and ground in one direction by a grinding rod made of glass, so that the kidney tissue smoothly passes through the stainless steel sieve, and the sieve is washed clean by flushing the sieve with HBSS at 4 ℃. The renal cortex tissue was passed through a 200 μm stainless steel cell sieve and a 100 μm cell sieve in the same manner. The undersize was collected and the oversize was gently scraped with a glass slide and transferred to a 50ml centrifuge tube. Gradient density centrifugation procedure was as follows: preparing 50ml of Percoll working solution with the concentration of 45%, averagely dividing the 50ml of Percoll working solution into two tubes, resuspending cell precipitates, centrifuging at 4 ℃ for 30min at an angle of 20000xg, after the centrifugation is finished, enabling the liquid to be divided into two layers, carefully extracting the suspension mixed solution at the lower layer, putting the suspension mixed solution into a 15ml centrifuge tube, adding HBSS, centrifuging at 760xg for 5min, adding HBSS, centrifuging again, repeating twice, and cleaning the Percoll. The obtained cells were cultured according to the renal tubular epithelial cell plate and culture protocol (3) of example 1.

As a result: after the cells are sieved, the number of dead cells accounts for the most part, the cells are seriously damaged by grinding before sieving, cell sap and nucleic acid are separated out to be sticky (figure 23A), and the activity is low (figure 23B).

From the state after the centrifugation process, there was a gray layer, which was supposed to be dead cell debris and debris (FIG. 23C). It can be seen that the total number of cells is large and the activity is still good after the percoll centrifugal separation procedure, but the cell amount after 3 days of the subsequent plating is shown in fig. 24, and the culture state of the subsequent plating plate is poor.

In conclusion, the method for separating and culturing the renal tubular epithelial cells can obtain high cell activity and high proliferation speed.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A method for separating and culturing renal tubular epithelial cells is characterized by comprising the following steps:

the first digestive enzyme comprises dispase and collagenase;

the second digestive enzyme is collagenase.

2. The isolation and culture method according to claim 1, wherein the collagenase is collagenase type II or collagenase type IV.

3. The isolation and culture method according to claim 1, wherein the concentration of the dispase in the first digestive enzyme is 0.75 to 1.5mg/mL, and the concentration of the collagenase is 1 to 2.5 mg/mL.

4. The isolation and culture method according to claim 1, wherein the concentration of the second digestive enzyme is 1 to 2.5 mg/mL.

5. The isolation and culture method according to claim 1, wherein the volume ratio of the collagenase to the dispase in the first digestive enzyme is 1: 0.5-2.

6. The separation and culture method according to claim 1, wherein the digestion time of the first digestive enzyme is 45-75 min, and the digestion time of the second digestive enzyme is 40-60 min.

7. The separation and culture method according to claim 1, wherein in the step S2, 2-3 mL of the first digestive enzyme is added per gram of the tissue fragment, and 0.5-0.1 mL of the second digestive enzyme is added per gram of the tissue fragment.

8. The isolation and culture method according to claim 1, wherein the tissue fragment of step S1 is 1-3 mm³。

9. The isolation and culture method according to claim 1, wherein the density of the cell seeding in step S3 is 0.5-1.5X 10⁶Per 75cm²。

10. The separation and culture method according to claim 1, wherein the conditions of the centrifugation in step S2 are: centrifuging at 800-1200 rpm for 4-10 min.