WO2022052338A1 - Preparation method for retinal ganglion cells - Google Patents

Abstract

A preparation method for Retinal ganglion cells (RGCs), improving related methods for separating and purifying the RGCs. Multiple experiments verify that the number of RGCs obtained by a single Retinal tissue on average is obviously higher than that of the original "two-step immunopanning method", and the purity of the separated RGCs is also obviously higher than that of the "two-step immunopanning method". Different batches of RGCs are immunofluorescently stained randomly by respectively using specificity marking antibodies of β-tubulin III and BRN3A. The results show that the purities of the RGCs marked by the two antibodies have no statistical difference, thereby further verifying that the "improved two-step immunopanning method" can obtain primary RGCs having relatively stable purity and yield.

Description

Preparation method of retinal ganglion cells

This application claims the priority of the Chinese patent application with the application number 202010961592.9 and the invention titled "Method for the Preparation of Retinal Ganglion Cells", which was filed with the China Patent Office on September 14, 2020, the entire contents of which are incorporated into this application by reference .

technical field

The present invention relates to the field of cells, in particular to a method for preparing retinal ganglion cells.

Background technique

It is estimated that the number of people with diabetes in the world will increase to 592 million by 2035. Diabetic retinopathy (DR) is a complex complication of diabetes, and its pathological features are progressive neurological dysfunction, accompanied by retinal microvascular degeneration, often leading to vision loss and even blindness. However, an increasing number of studies have shown that RGCs damage occurs early in retinopathy, possibly prior to visible retinal vasculopathy. Endoplasmic reticulum stress and mitochondrial oxidative stress further accelerated the apoptosis of RGCs in diabetic retinopathy.

Glaucoma is an eye disease characterized by vision loss and visual field defects. The typical pathological features are changes of the optic nerve head and reduction of RGCs. In addition to age, elevated intraocular pressure is considered to be the main reason for the decrease in the number of RGCs. In the pathological changes caused by glaucoma, the axons of RGCs are the first structures to be destroyed. The degeneration of RGCs and the resulting optic nerve damage can lead to progressive loss of vision and eventually blindness. Currently, there is no curative treatment for glaucoma. Because glaucoma is a complex multifactorial disease, its development process is variable. Although some patients have well-controlled intraocular pressure, their visual acuity continues to decline. Therefore, secondary degeneration of RGCs is thought to play an important role in the entire pathological process.

Therefore, how to better study these difficult ophthalmological problems and how to better promote the progress of clinical treatment are questions that ophthalmologists around the world are thinking about. This requires in-depth research starting from cytology, to better study the characteristics of RGCs, and to screen new and more effective drugs in a more targeted manner. At present, the "two-step immunodisking method" created by Barres is used for the purification of RGCs. However, primary retinal ganglion cells, as one of the central neurons, face many challenges in extraction, purification and culture, such as low purity, small number of cells, and difficulty in in vitro culture. Therefore, how to better extract, purify and culture primary RGCs is an urgent problem to be solved in the basic research of ophthalmology.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a method for preparing retinal ganglion cells. The present invention improves the original "two-step immune discization method", and obtains primary RGCs with high purity and high yield.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The preparation method of retinal ganglion cells provided by the present invention comprises the following steps:

Step A, obtaining retinal cell suspension;

Step B, prepare a negative screening carrier and a positive screening carrier; the negative screening carrier is coated with goat anti-rabbit IgG (H+L) secondary antibody, and the positive screening carrier is coated with goat anti-mouse IgG+IgM (H+L) )Secondary Antibodies;

Step C. Coat the positive screening vector with primary antibody (labeling retinal ganglion cells and non-retinal ganglion cells);

Step D. Mix the rabbit anti-rat macrophage polyclonal antibody (CedarlaneCLAD51240) with the retinal cell suspension prepared in step A, incubate and centrifuge to obtain the antibody-labeled retinal cell suspension;

Step E, take the retinal cells obtained in step D and resuspend, and then screen the negative screening vector obtained in step B, collect the cell suspension, and then screen the positive screening vector obtained in step B, remove the cells containing The cell suspension of non-retinal ganglion cells is the supernatant, the cells are collected, cultured and purified;

Wherein, the order of step A and step B is in no particular order;

The collection of cells in step E is to separate the retinal cells by physical pipetting without using digestive enzymes when separating the retinal ganglion cells bound to the positive screening vector.

Depending on the principle of use, we use an incubation time of 10 minutes. In the process of positive discization, because the bottom of the petri dish is coated with CD90 antibody, the antibody binds to the specific antigen on the cell surface and causes the RGCs to settle into the petri dish. The time at the bottom is shorter than that of other non-RGCs. Therefore, the "improved two-step immunoplating method" utilizes the time difference of sedimentation between RGCs and non-RGCs to separate and purify RGCs, shorten the entire purification process, and obtain RGCs with high purity and high yield. The RGCs cell yield (203000±6173/retina) of the “improved two-step immunodiscination method” was much higher than that of the original “two-step immunodiscination method” (18890±484.4/retina). Moreover, when the RGCs settled to the bottom of the positive screening petri dish were separated, the separation method of the original "two-step immunodisking method", which was washed with a large amount of D-PBS and then digested with digestive enzymes, was not used. During the D-PBS rinsing process, a large number of RGCs were lost, which greatly reduced the yield of cells, and the activity of retinal ganglion cells was greatly reduced after enzymatic digestion, and some cells died directly. In the "improved two-step immunodiscination method", which takes advantage of the fact that the CD90 antibody does not bind to the surface antigen of RGCs very closely, the physical method of gentle pipetting with D-PBS makes the antibody coated on the bottom of the dish interact with the cells. The antigens are separated, so that for the RGCs with weak vitality in vitro, the cell activity can be maintained to the maximum extent, and the influence of external factors can be reduced, and the isolated RGCs can be close to the original vitality state to the greatest extent. .

In some embodiments of the present invention, step A employs a digestive enzyme with milder digestive properties, the digestive enzyme including papain.

In some specific embodiments of the present invention, the incubation in step D to obtain the retinal cell suspension is incubation at 18-25° C. for 10 min.

In some specific embodiments of the present invention, the primary antibody in step C is a mouse anti-rat CD90 antibody.

In some specific embodiments of the present invention, step A, step D, step E use high oogen solution, low oogen solution and/or discization buffer as the solution;

In mg/mg/mL/μL, the weight-to-volume ratio of each component in the high ovogen solution is: bovine serum albumin: trypsin inhibitor: Dulbecco's phosphate buffer: 1N NaOH=600:600:20 :150;

In mg/mg/mL/μL, the weight-to-volume ratio of each component in the low ovogen solution is: bovine serum albumin: trypsin inhibitor: Dulbecco's phosphate buffer: 1N NaOH=300:300:20 :100.

In some specific embodiments of the present invention, the culture described in step E adopts RGCs medium, which consists of the following components:

In some specific embodiments of the present invention, the DMEM-SATO basal medium consists of the following components:

In some specific embodiments of the present invention, the centrifugation in step D is centrifugation at 18-25° C. and 900 rpm for 5 min.

The present invention improves the relevant methods for the separation and purification of RGCs. It has been confirmed by multiple experiments that the number of RGCs obtained by an average single retinal tissue is significantly higher than the original "two-step immune discization method", and the purity of the isolated RGCs is also significantly higher than that of " Two-step immunodisking method". Immunofluorescence staining of different batches of RGCs was performed using specific labeled antibodies β-tubulin III and BRN3A. The results showed that there was no statistical difference in the purity of the two antibody-labeled RGCs, which further verified that the "improved two-step immunoplating method" could obtain primary RGCs with relatively stable purity and yield. This study lays a cytological basis for large-scale and in-depth exploration of the mechanism of vision loss caused by necrosis and apoptosis of RGCs.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the preparation of positive and negative culture dishes;

Figure 2 shows a coated cell culture plate;

Figure 3 shows the isolated retina;

Figure 4 shows the preparation of primary retinal cell suspension;

Figure 5 shows the preparation of low ovogen solution and high ovogen solution;

Figure 6 shows that retinal cells bind to specific antibodies;

Figure 7 shows selection of cells using negative and positive dishes;

Figure 8 shows that cells were resuspended in RGCs medium, and plated after cell counting;

Figure 9 shows the isolation of primary SD rat RGCs by the "improved two-step immunodiscination method"; the cells that emit green fluorescence in Figure 9(A) are RGCs labeled with β-tubulin III; the red fluorescence is BRN3A-specific Antibody-labeled RGCs; blue are DAPI-labeled nuclei. Scale bar: 20 μm; Figure 9(B) shows that β-tubulin III and BRN3A specifically label RGCs, respectively, and there is no statistical difference in cell purity between the two. The purity is relatively stable, and the data are expressed as mean±standard error; β-tubulin III: 90.06±0.1696%; BRN3A: 88.51±0.5859% (n=3; ns: P=0.0632>0.05);

Figure 10 shows the average yield of primary SD rat RGCs isolated by different immunodiscination methods; data are expressed as mean ± standard error. "Two-step immunoplating method": 18890±484.4 cells/retina; "Improved two-step immunoplating method": 203000±6173 cells/retina (n=3; ***: P<0.0001);

Figure 11 shows the comparison of the purity of primary SD rat RGCs isolated by different immune separation methods; wherein, Figure 11 (A) green fluorescence is RGCs labeled with β-tubulin III, blue fluorescence is DAPI-stained nuclei, scale bar: 20 μm; Figure 11(B) Data are expressed as mean ± standard error; "two-step immunoplating method" RGCs purity: 72.29 ± 1.025%; "improved two-step immunoplating method": 90.06 ± 0.1696% (n=3; ***:P<0.0001).

detailed description

The present invention discloses a preparation method of retinal ganglion cells, and those skilled in the art can learn from the content of this paper and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

RGCs are neurons located in the innermost ganglion cell layer of the retina and are composed of multipolar ganglion cells. Dendrites are mainly associated with bipolar cells, but also laterally with podocytes. RGCs converge on the slender axons in the brain to form the optic nerve, which is an important channel for transmitting visual signals. They play a crucial role in transmitting visual information from photoreceptors to the brain, and in the process of glaucoma, diabetic retinopathy, and optic nerve damage caused by trauma, accelerate the necrosis and apoptosis of RGCs, resulting in visual damage that Vision loss. Therefore, deepening the research on RGCs and exploring the mechanism of damage and apoptosis will help to further deepen the understanding of the pathological process of glaucoma and diabetes, delay the loss of vision, and improve the prognosis of patients. Therefore, related research on RGCs is a bridge between basic pathological research and clinical treatment of patients. However, as a kind of central neurons, RGCs are very sensitive to the external environment and are prone to disintegration and apoptosis in vitro. Moreover, the number of RGCs obtained by the original "two-step immune discization method" is small and the purity is limited, which cannot meet a large number of cell experiments, which is not conducive to our research on vision loss caused by eye diseases such as glaucoma and diabetes. The basic research of RGCs-related diseases has caused great obstacles, so it is more urgent to explore a more stable and efficient method for purifying and culturing primary RGCs.

In our study, we have innovated in the principle of coating antibody, and improved the method of "two-step immunodiscination" to extract RGCs, so that the whole process time is shorter, more cells are obtained, and the cell purity is higher. . The purity (90.06±0.1696%) of the RGCs purified by the "improved two-step immunoplating method" was significantly higher than that of the two-step immunostaining method (72.29±1.025%). In the original "two-step immunoplatenization method", the cell suspension was incubated for 45 minutes during positive selection. Through our extensive research, we found that during the 45-minute positive screening process, with the extension of the incubation time, endothelial cells, macrophages and microglia will settle to the bottom of the negative screening dishes due to gravity during this time period. , and due to the attractive interaction between cells, the presence of heterocytic cells is increased, resulting in an increase in the number of non-RGCs, which greatly reduces the purity. In our "improved two-step immunodiscination method", according to the principle of use, we use an incubation time of 10 minutes. Binding to specific antigens on the cell surface allows RGCs to settle to the bottom of the dish in less time than other non-RGCs. Therefore, the "improved two-step immunoplating method" utilizes the time difference of sedimentation between RGCs and non-RGCs to separate and purify RGCs, shorten the entire purification process, and obtain RGCs with high purity and high yield. The RGCs cell yield (203000±6173/retina) of the "improved two-step immunoplating method" was much higher than that of the original "two-step immunoplating method" (18890±484.4/retina). Moreover, when the RGCs settled to the bottom of the positive screening petri dish were separated, the separation method of the original "two-step immunodisking method", which was washed with a large amount of D-PBS and then digested with digestive enzymes, was not used. During the D-PBS rinsing process, a large number of RGCs were lost, which greatly reduced the yield of cells, and the activity of retinal ganglion cells was greatly reduced after enzymatic digestion, and some cells died directly. In the "improved two-step immunoplatenization method", which takes advantage of the fact that CD90 is not very tightly bound to the surface antigen of RGCs, it uses the physical method of gentle pipetting with D-PBS to make the antibody coated on the bottom of the dish and the antigen of the cell. For RGCs with weak in vitro vitality, the cell activity can be maintained to the maximum extent, and the influence of external factors can be reduced, and the isolated RGCs can be close to the original vitality state to the greatest extent.

In conclusion, the purity of the RGCs obtained by the "improved two-step immunoplating method" was about 90%, which was significantly higher than that of the original "two-step immunoplating method", which was 72.29±1.025%; and in the obtained cells In terms of yield, the "improved two-step immunoplating method" (203000±6173/retina) was about 10 times that of the original "two-step immunodiscination method" (18890±484.4/retina). Therefore, the "improved two-step immunoplating method" is obviously superior to the original "two-step immunoplating method", which makes it possible to obtain high-purity and high-yield primary SD rat RGCs, which are suitable for glaucoma and diabetic retinopathy. The research on the mechanism of vision loss caused by other diseases has laid a solid cytological foundation.

The raw materials and reagents used in the preparation method of retinal ganglion cells provided by the present invention can be purchased from the market.

Animals: Primary retinal ganglion cells were extracted from SD rat pups born at 48-72 h and purchased from the Experimental Animal Center of Zhengzhou University. All animals were sacrificed by cervical dislocation.

Reagent preparation table for RGCs cell culture:

Table 1 Composition of RGCs medium

reagent added amount DMEM-SATO basal medium (Table 2) 20mL Forskolin (4.2mg/mL) (Table 3) 20μL Brain-derived neurotrophic factor (50μg/mL) 20μL Ciliary neurotrophic factor (10μg/mL) 20μL

Table 2 Composition of DMEM-SATO basal medium

reagent added amount Neural Basal Medium (Gibco 21103-049) 9.2mL DMEM medium (Gibco 11960-044) 9.2mL Insulin (Absin abs9169-25mg) (Table 4) 200μL Sodium Pyruvate (Gibco 11360-070) 200μL Penicillin-streptomycin (Gibco 15140-122) 200μL L-Glutamine (Gibco 25030-081) 200μL NS21 Additive (50×) (R&D Systems AR008) 400μL Thyroxine (T3) (4 μg/mL; Sigma-Aldrich T6397) (Table 5) 200μL N-Acetyl-L-cysteine (5 mg/mL; Sigma-Aldrich A8199) (Table 6) 20μL SATO additive (100×) (Table 7) 200μL

The above reagents were mixed and sterilized through a 0.22 micron filter

Table 3 Forskolin preparation table (4.2mg/mL)

reagent added amount Forskolin (Sigma-Aldrich F6886) 10mg Dimethyl sulfoxide (Solarbio D8371) 2.4mL

Mix well, filter sterilize through a 0.22-μm Teflon filter, aliquot 100 μL, and store at -20°C.

Table 4 Insulin preparation table (0.5mg/mL; Absin abs9169-25mg)

reagent added amount

Insulin (0.5mg/mL; Absin abs9169-25mg) 2mg Sterilized water 4mL 1.0N HCl 20μL

Mix well on ice and filter sterilize through a 0.22 μm filter. Store at 4°C.

Table 5 Thyroxine (T3) preparation table (8mg/mL)

reagent added amount Thyroxine sodium salt (T3; Sigma-Aldrich T6397) 1.6mg 0.1N NaOH 200μL

Prepare 4 μL/mL thyroxine (T3) stock solution: Add 5 μL of T3 solution (8 mg/ml) to 10 mL of phosphate buffered saline (D-PBS; Gibco 14287). Filter sterilize through a 0.22 micron filter, aliquot into 500 μL aliquots, and store at -20°C.

Table 6 N-acetyl-L-cysteine preparation table (5mg/mL)

Mix well, filter sterilize through a 0.22 μm filter, aliquot into 100 μL aliquots, and store at -20°C.

Table 7 SATO (the abbreviation is the English abbreviation of the following reagents) additive preparation table (100×)

reagent added amount Putrescine (Sigma-Aldrich P5780) 32mg Bovine Serum Albumin (BSA) (Sigma-Aldrich A4161) 200mg Sodium selenite stock solution (Table 8) 200μL Transferrin (Sigma-Aldrich T1147) 200mg Progesterone stock solution (Table 9) 5μL

Add 20 ml of DMEM to the above reagent, stir gently, sterilize with a 0.22 micron filter, aliquot into 500 microliter aliquots, and store at -20°C.

Table 8 Preparation table of sodium selenite stock solution

reagent added amount Sodium Selenite (Sigma-Aldrich S5261) 1mg DMEM (Gibco 11960-044) 2.5mL 1N NaOH 2.5μL

Mix gently and set aside.

Table 9 Progesterone stock solution preparation table

reagent added amount Progesterone (Sigma-Aldrich P8783) 1mg 75% ethanol 40μL

Mix gently and set aside.

Reagent preparation table used in separation and purification:

Table 10 0.2% bovine serum albumin solution (0.2% BSA) preparation table

Mix well, sterilize with a 0.22 micron filter, and store at -20°C.

Table 11 DNase preparation table

reagent added amount DNase (Worthington LS002007) 3mg Balanced Salt Solution (EBSS) (Solarbio H2040) 2mL

Prepare on ice, sterilize with a 0.22 micron filter, and store at -20°C.

Table 12 "High Ovogen Solution" Preparation Table

Mix well and filter sterilize through a 0.22 micron filter. Store at -20°C.

Table 13 "Low Ovogen Solution" Preparation Table

Mix well and filter sterilize through a 0.22 micron filter. Store at -20°C.

Table 14 Preparation of discization buffer

Table 15 Tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl) (50mM, pH9.5)

reagent added amount Tris(hydroxymethyl)aminomethane (Tris) 12.1g distilled water 200ml

Mix thoroughly, adjust the pH to 9.5 with dilute hydrochloric acid, and use after high temperature and high pressure sterilization.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Extraction of RGCs from 20-25 SD rat pups within 48-72 h after birth

first day:

1. Prepare petri dishes for positive screening and negative screening (Figure 1)

1.1 Prepare two 50ml centrifuge tubes marked as "negative screening", add 40ml of 50mM Tris-HCl (Tris-HCl buffer) (pH 9.5) and 120μL of goat antibody to each centrifuge tube Rabbit immunoglobulin (H+L) (Jackson 111-005-003), mix well and set aside.

1.2 Prepare four Petri dishes for negative screening, marked as A1, A2, B1, B2;

Add 20 mL of the mixture of antibody and Tris-HCl to each 15cm petri dish, mix gently, seal with sterilized parafilm, and place in a 4°C refrigerator overnight for later use.

1.3 Take a 50 ml centrifuge tube labeled "positive screening", add 20 ml of 50 mM Tris-HCl (pH 9.5), 60 μl of goat anti-mouse immunoglobulin IgM (H+L) (Jackson Corporation 115-005- 044), mix gently.

1.4 Prepare 2 10cm sterile petri dishes, labeled A and B. Add 10 ml of the mixed solution of "positive screening" antibody and Tris-HCl to each petri dish, mix gently, seal with sterilized parafilm, and store in a refrigerator at 4°C overnight for later use.

2. Coating the Cell Culture Plate (Figure 2)

2.1 According to the needs of grouping, select the number of 24-well plates, and add 500 μL of polylysine solution (Sigma-Aldrich P4707-50ML) to each 24-well plate to make it evenly cover the bottom of the plate. Allow to coat at room temperature for at least 2 hours.

2.2 Use neural basal medium (Gibco 21103-049) to dilute laminin (Gibco 23017-015) to a concentration of 10 μg/ml, and mix well for later use. The polylysine in the wells was recovered, and then diluted laminin was added, 500 μL per well. It was then placed in a 37°C incubator overnight.

the next day:

1. Coat positive screening dishes with primary antibodies

1.1 Take 13.5ml of D-PBS (HyClone SH30264.01) and 1.5ml of 0.2% BSA (SigmaAldrich A1933-25G) solution, then add 100μL of mouse anti-rat CD90 antibody (Abd serotec MCA04G) by pipetting gently, mix Even, spare.

1.2 Rinse the 2 10cm "positive selection" dishes prepared on day 1 3 times with D-PBS. Add 7.5 mL of mouse anti-rat CD90 antibody solution to each petri dish and incubate at room temperature (18°C-25°C) for 2 hours.

2. Detach the retina

2.1 Steps to separate retinas (Figure 3)

2.1.1 The suckling mice of SD rats at 48-72 h after birth were killed by cervical dislocation, placed in 75% alcohol for 3 minutes, and then their eyeballs were quickly removed and placed in a petri dish containing cold D-PBS in advance. Retinal tissue in the eyeball was isolated using a dissecting microscope.

First, fix the eyeball with micro tweezers, cut a small hole with micro scissors, and start cutting along the corneoscleral limbus. The lens and cornea at the front of the eye are removed. Second, fix the caudal end of the optic nerve directly in front of the eyeball with microtweezers and gently squeeze the eyeball in the opposite direction with another pair of microtweezers to float the retina out of the sclera. Finally, the vascular membrane attached to the retina was gently pulled and removed, and the retina was transferred to a 1 mL EP tube containing cold D-PBS for preservation.

3. Preparation of Primary Retinal Cell Suspension (Figure 4)

3.1 Prepare three 15mL centrifuge tubes as described below

3.1.1 Take a centrifuge tube and add 5ml of D-PBS and 70μL of papain (Worthington Biochemical LS003126) labeled as "papain tube". After mixing, water bath at 37°C for 5 minutes.

3.1.2 Take a 15mL centrifuge tube and add 9mL of D-PBS, marked as "low egg stock solution tube", and add 5mL of D-PBS to another centrifuge tube and mark it as "high egg stock solution tube" for use.

3.2 Weigh 1 mg of L-cysteine and add it to the "papain tube" and add 5 μL 1N NaOH to adjust the pH.

3.3 In the "papain tube", add 190 μL DNase (Worthington LS002007), pipetting and mixing, and then use a 0.22 μm (Millipore SLGV033RS) filter to filter and sterilize.

3.4 Transfer the prepared retinal tissue to a filter-sterilized papain tube using a 1 mL pipette and heat in a 37-degree water bath for 20 minutes to digest the tissue.

3.5 Preparation of "low egg stock solution" and "high egg stock solution" (Figure 5)

Add 1mL of "low egg stock solution" and 2μL of 1N NaOH to the centrifuge tube marked with "low egg stock solution" and mix well; add 1mL of "high egg stock solution" and 2μL of 1N to the centrifuge tube marked with "high egg stock solution" NaOH was mixed and used for later use.

3.6 After 20 minutes, aspirate the supernatant in the "papain tube" and drop 4 ml of "low egg stock solution" into the tube. Let stand for 3 minutes to stop papain digestion.

4 Retinal cells bind to specific antibodies (Figure 6)

4.1 Add 90 μL of rabbit anti-rat macrophage polyclonal antibody (Cedarlane CLAD51240) and mix it with the remaining 6 mL of “low egg stock solution” for use.

4.2 Gently pipet the "low egg stock solution", and add 2 ml of the mixed solution of the "low egg stock solution" and rabbit anti-rat macrophage antibody to the digested retinal tissue. Using a 3 ml disposable pipette, triturate the retina 8-10 times and let the solution sit for 1 min.

4.3 After 1 minute, transfer the supernatant to a new 15 ml tube. Add 1 ml of "low egg stock solution + anti-macrophage antibody" (90μL rabbit anti-rat macrophage polyclonal antibody (Cedarlane CLAD51240) to the original test tube again and mix it with the remaining 6 ml of "low egg stock solution" to prepare " Low egg stock solution + anti-macrophage antibody mixture") mix the solution, triturate the retina 8-10 times, and let the solution stand for 1 minute. Repeat the same process until all the mixed solution of "low egg stock solution + anti-macrophage antibody" is used up.

4.4 Incubate the retinal cell suspension obtained after grinding for another 10 minutes at room temperature, so that the rabbit anti-rat macrophage antibody binds to the specific cell antigen epitope.

4.5 Centrifuge the retinal cell suspension at 900 rpm for 5 min at room temperature (the room temperature in the present invention is 18-25° C.).

5. Screening of retinal ganglion cells using coated negative and positive dishes (Figure 7)

5.1 Prepare buffer:

36mL of D-PBS, 4mL of 0.2% BSA, and 400μL of insulin solution (Absin abs9169-25mg) were placed in a 50mL centrifuge tube, thoroughly mixed, and the centrifuged cells were resuspended in buffer and pipetted evenly.

5.2 Use a 40 μm nylon mesh to filter the resuspended single cell suspension to remove undigested tissue.

5.3 Transfer the filtered cell suspension to two 15 cm negative selection petri dishes labeled A1 and B1. Let stand at room temperature for 25 minutes.

5.4 After 25 minutes, the cells were transferred to two other 15cm negative screening dishes marked A2 and B2 respectively, and allowed to stand at room temperature for 30 minutes. The macrophages and microglia in the cell suspension were removed by the above negative screening. cells and endothelial cells.

5.5 Discard the mouse anti-rat CD90 antibody solution in the two 10cm positive screening dishes, and after rinsing 3 times with D-PBS, transfer the cell suspension that passed the negative screening to the positive screening dishes marked A and B, respectively , let stand at room temperature for 10 minutes.

5.6 After 10 minutes, remove the supernatant from the 10 cm positive screening dish with a disposable 3 mL pipette. Examine the dish under the microscope to ensure that only cells remain. Then an appropriate amount of D-PBS was added to rinse the 10 cm dish to wash off the cells adsorbed on the bottom of the dish. Check the dish under the microscope to make sure there are no cells attached to the bottom of the dish.

5.7 Transfer the cell solution in two 10cm dishes to two 15ml centrifuge tubes and label them A, B centrifuge tubes. Then centrifuge at 900 rpm for 5 minutes at room temperature (18-25°C).

6. Resuspend cells in RGCs medium and count using a cell counter (Figure 8)

6.1 After resuspending the RGCs, adjust the cell density to 10 ⁵ /mL.

6.2 Rinse the coated 24-well plate 2-3 times with D-PBS, and spread the RGCs in the 24-well plate at a density of 10 ⁵ /mL. Culture in an incubator at 37 °C, 5% _CO and 95% air.

7. Further purification of RGCs

7.1 In order to better reflect the characteristics of cells and meet the needs of the experiment, RGCs need to be further purified.

7.2 After one day, replace with a new medium (as shown in Table 1) to remove the dead and bad RGCs. After the above treatment, the cell purity can reach about 90% (detection method: the cell purity detection adopts the Two specific markers of RGCs, randomly fluorescently stained RGCs extracted from different batches, dividing the number of cells stained with specific markers by the number of cells stained with DAPI multiplied by 100%, as a measure of cell purity . Detection conditions: room temperature 18 to 25 ℃), to meet the needs of subsequent experiments.

Example 2 Immunofluorescence staining

Twenty-four hours after plating, the obtained cells were subjected to immunofluorescence staining with specific markers to determine cell purity. After 24 hours of plating, the cell culture medium of the RGCs was discarded, washed three times with PBS (Solarbio P1020-500ml), and fixed with immunofixation solution (Beyotime P0098-100mL) for 15min at room temperature. Immunostaining Wash Buffer (Beyotime P0106), washed 3 times for 10 minutes each. After rinsing, block with immunostaining blocking solution (Beyotime P0102) for 1 h at room temperature. This was followed by overnight incubation at 4 degrees with anti-β-tubulin III antibody (1:500, Abcam ab7751) or anti-BRN3A antibody (1:500, bs-3669R). Wash 3 times with Immunostaining Wash Buffer (Beyotime P0106) for 5 min each. Then in the dark, the corresponding secondary antibodies (donkey anti-mouse IgG H&L Alexa Fluor 488, 1:1000 dilution, Abcam ab150105; donkey anti-rabbit IgG H&L Alexa Fluor 647, 1:1000 dilution, Abcam, ab150075) were incubated at room temperature Incubate for 2 hours. Then, wash 3 times with Immunostaining Wash Solution (Beyotime P0106) for 5 min each. Nuclei were counterstained with DAPI (Beyotime C1005) for 5 minutes at room temperature, and then rinsed with immunostaining wash solution for 3 times, 5 minutes each. After rinsing, an appropriate amount of anti-immunofluorescence quencher (Abcam ab104135) was added dropwise. Using OLYMPUS fluorescence microscope software, Alexa Fluor and DAPI were excited at 488nm, 568nm, and 358nm, respectively, and a field of view was randomly selected to take pictures under a fluorescence microscope at 400X. Each group was repeated 3 times, and the obtained pictures were analyzed using Image J software. The purity and yield of cells were calculated and compared with the original immunoisolation method.

Example of effect

1.1 Purity of primary SD rat retinal ganglion cells extracted by "improved two-step immunodiscination method"

Different batches of primary RGCs obtained were randomly labeled with different specific markers (Different makers) β-tubulin III or BRN3A for RGCs (Figure 9). The results showed that the cell purity of β-tubulin III labeled RGCs (Purity of RGCs): 90.06±0.1696%; the cell purity of BRN3A-labeled RGCs was 88.51±0.5859%, and there was no statistical difference between the two. This result proves that "Improved two-step immunopanning method" can obtain RGCs with higher cell purity and more stable.

Table 16 Immunofluorescence staining of different specific markers of RGCs to compare the purity of cells extracted from different batches

P=0.0632>0.05, which indicated that the use of different specific markers for random detection and the "improved two-step immunoplating method" could obtain primary RGCs with high purity and stable percentage (the experiment was repeated three times).

1.2 Yield of primary SD rat retinal ganglion cells obtained by different immune separation and purification methods

The yield of cells obtained by the original "Two-step immunopanning method" and the "Improved two-step immunopanning method" were carried out. The yields of primary SD rat retinal ganglion cells obtained by two different methods were counted and quantitatively analyzed. There was a huge difference in the yield of RGCs between the two methods. The average yield of the "two-step immunoplating method" was 18890±484.4 cells/retina, and the yield of the "modified two-step immunodiscination method" was about 203000±6173 cells/retina. The yield of the "improved two-step immunoplatenization method" was significantly higher than that of the original "two-step immunoplatenization method" (Fig. 10).

Table 17 Comparison of yields of primary RGCs cells obtained by two different immunoisolation and purification methods

P<0.0001, the yields of primary RGCs cells obtained by two different immune separation and purification methods were significantly different, and the "improved two-step immunoplating method" was much higher than the "two-step immunoplating method" (the experiment was repeated three times) ).

1.3 Purity of primary SD rat retinal ganglion cells obtained by different immunoisolation methods.

From the results in 1.1, as a specific marker of RGCs, β-tubulin III or BRN3A had no statistical difference in the determination of the purity of labeled RGCs. Therefore, we only used β-tubulin III for labeling, and then compared the cell purity obtained by different immunoplating methods. The purity of RGCs isolated by "two-step immunoplating method" was 72.29±1.025%, which was significantly lower than 90.06±0.1696% of "improved two-step immunoplating method" (Fig. 11), and there was a statistical difference (P<0.0001) .

Table 18 Comparison of the purity of RGCs obtained by two different immunoisolation and purification methods

P<0.0001, there are significant differences in the purity of primary RGCs obtained by the two different immune separation and purification methods. ).

The preparation method of retinal ganglion cells provided by the present invention has been described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (8)

A method for preparing retinal ganglion cells, comprising the steps of: Step A, obtaining retinal cell suspension; Step B, prepare a negative screening carrier and a positive screening carrier; the negative screening carrier is coated with goat anti-rabbit IgG (H+L) secondary antibody, and the positive screening carrier is coated with goat anti-mouse IgG+IgM (H+L) )Secondary Antibodies; Step C, coating the positive screening vector with primary antibody; Step D, mixing the rabbit anti-rat macrophage polyclonal antibody with the retinal cell suspension prepared in step A, incubating, and centrifuging to obtain retinal cells; Step E, take the retinal cells obtained in step D and resuspend, after the screening of the negative screening vector obtained in step B, collect the cell suspension, and then screen the positive screening vector obtained in step B, remove the upper Serum, collect cells, culture, purify; Wherein, the order of step A and step B is in no particular order; The cells collected in step E did not use digestive enzymes, and the retinal cells were separated by pipetting. The preparation method according to claim 1, characterized in that, in step A, a digestive enzyme is used to obtain a retinal cell suspension; and the digestive enzyme comprises papaya digestive enzyme. The preparation method according to claim 1 or 2, wherein the incubation in step D is incubation at 18-25° C. for 10 min. The preparation method according to any one of claims 1 to 3, wherein the primary antibody in step C is a mouse anti-rat CD90 antibody. The preparation method according to any one of claims 1 to 4, characterized in that, in Step A, Step D, and Step E, a high ovogen solution and/or a low oogen solution and a discization buffer are used as solutions; In mg/mg/mL/μL, the weight-to-volume ratio of each component in the high ovogen solution is: bovine serum albumin: trypsin inhibitor: Dulbecco's phosphate buffer: 1 N NaOH=600:600: 20:150; In mg/mg/mL/μL, the weight-to-volume ratio of each component in the low ovogen solution is: bovine serum albumin: trypsin inhibitor: Dulbecco's phosphate buffer: 1 N NaOH=300:300: 20:100. The preparation method according to any one of claims 1 to 5, wherein the culture in step E adopts RGCs medium, which consists of the following components:

The preparation method of claim 6, wherein the DMEM-SATO basal medium consists of the following components:

The preparation method according to any one of claims 1 to 7, wherein the centrifugation in step D is centrifugation at 18-25° C. and 900 rpm for 5 min.

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