CN112251408B - Preparation method of retinal ganglion cells - Google Patents

Abstract

The invention relates to the field of cells, in particular to a preparation method of Retinal Ganglion Cells (RGCs). The invention improves the related method for separating and purifying the RGCs, and multiple experiments prove that the number of the RGCs obtained by averaging a single retinal tissue is obviously higher than that of the original two-step immunodomination method, and the purity of the separated RGCs is also obviously higher than that of the two-step immunodomination method. Different batches of RGCs were immunofluorescent stained using the specific marker antibody β -tubulin III, BRN 3A. The results show that the purity of the RGCs marked by the two antibodies has no statistical difference, and further verify that the improved two-step immune disrotatory method can obtain primary RGCs with relatively stable purity and yield. The research lays a cytological foundation for large-scale deep research on the mechanism of vision deterioration caused by RGCs necrosis and apoptosis.

Description

Preparation method of retinal ganglion cells

Technical Field

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

Background

It is estimated that the number of diabetics will increase to 5.92 million worldwide by 2035 years. Diabetic Retinopathy (DR) is a complex complication of diabetes mellitus, which is pathologically characterized by progressive neurological dysfunction with retinal microvascular degeneration, often resulting in vision loss, even blindness. However, increasing research has shown that RGCs damage occurs early in retinopathy, possibly before visible retinal vasculopathy. Endoplasmic reticulum stress and mitochondrial oxidative stress further accelerate apoptosis of RGCs in diabetic retinopathy.

Glaucoma is an ophthalmic disease characterized primarily by vision loss and visual field loss. Typical pathological features are changes in the optic nerve head and a decrease in RGCs. In addition to age, elevated intraocular pressure is believed to be the primary cause of the reduction in the number of RGCs. In the pathological changes caused by glaucoma, the axons of RGCs are the first structures to be destroyed. Degeneration of RGCs and the resulting optic nerve damage can lead to gradual loss of vision and ultimately blindness. At present, there is no curative treatment for glaucoma. Since glaucoma is a complex multifactorial disease, its progression is variable. Although intraocular pressure control is good in some patients, vision continues to decline. Thus, secondary RGCs degeneration is thought to play an important role throughout the pathological process.

Therefore, how to better study these ophthalmic problems and how to better promote the progress of clinical treatment is a problem that ophthalmologists think all over the world. It is necessary to take in-depth studies from cytology to better study the characteristics of RGCs and to screen new, more effective drugs more specifically. Currently, Barres 'two-step immunoplate' is used for RGCs purification. However, primary retinal ganglion cells, which are one of the central neurons, face many challenges such as low purity, small cell number, and difficulty in vitro culture in terms of extraction, purification, and culture. Therefore, how to better extract, purify and culture primary RGCs is a problem to be solved urgently in basic research of ophthalmology.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing retinal ganglion cells. The invention improves the original 'two-step immunization disk method' and obtains the primary RGCs with high purity and high yield.

In order to achieve the above object, the present invention provides the following technical solutions:

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

step A, obtaining a retinal cell suspension;

step B, preparing and obtaining a negative screening vector and a positive screening vector; the negative screening vector is coated with a goat anti-rabbit IgG (H + L) secondary antibody, and the positive screening vector is coated with a goat anti-mouse IgG + IgM (H + L) secondary antibody;

step C, coating the positive screening carrier with primary antibody (marking retinal ganglion cells and non-retinal ganglion cells);

step D, mixing a rabbit anti-rat macrophage polyclonal antibody (Cedarlane CLAD51240) with the retinal cell suspension prepared in the step A, incubating and centrifuging to obtain an antibody-labeled retinal cell suspension;

step E, taking the retinal cells prepared in the step D for re-suspension, screening the negative screening carrier prepared in the step B, collecting cell suspension, screening the positive screening carrier prepared in the step B, removing the cell suspension containing the non-retinal ganglion cells, namely supernatant, collecting the cells, culturing and purifying;

wherein, the sequence of the step A and the step B is not sequential;

and E, collecting the cells, namely separating the retinal ganglion cells bound with the positive screening carrier by adopting a physical blowing and beating mode without using digestive enzyme.

According to the principle of use, we used 10 minutes incubation time, during positive dishing, since the bottom of the dish was coated with CD90 antibody, which binds to the cell surface specific antigen, allowing RGCs to settle to the bottom of the dish in less time than other non-RGCs. Therefore, the improved two-step immune disrotatory method is to separate and purify the RGCs by utilizing the time difference of sedimentation between the RGCs and non-RGCs, shortens the whole purification process and can obtain the RGCs with high purity and high yield. The cell yield (203000 +/-6173/retina) of the RGCs of the improved two-step immune disketting method is far higher than that of the original two-step immune disketting method (18890 +/-484.4/retina). Moreover, when separating the RGCs settled to the bottom of the positive screening culture dish, a separation method of digesting by digestive enzyme after rinsing by a large amount of D-PBS in the original 'two-step immunization disk method' is not used, and the method can cause a large amount of RGCs to be lost in the rinsing process of the D-PBS, so that the yield of cells is greatly reduced, the activity of retinal ganglion cells is greatly reduced after the enzymatic digestion, and partial cells directly die. The improved two-step immune disklization method utilizes the characteristic that the combination of a CD90 antibody and RGCs surface antigen is not very tight, and uses a physical method of gentle blowing and beating by D-PBS to separate the antibody coated at the bottom of a culture dish from the antigen of cells, so that the activity of the cells of the RGCs with weak in vitro life activity can be kept to the maximum extent, the influence of external factors is reduced, and the separated RGCs can be close to the original activity state to the maximum extent.

In some embodiments of the invention, step a employs digestive enzymes having softer digestive characteristics, including papain.

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

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

In some embodiments of the invention, step a, step D, step E employ a high oogenesis solution, a low oogenesis solution and/or a panning buffer as a solution;

the weight volume ratio of each component in the homoovarial solution is as follows in mg/mg/mL/muL: bovine serum albumin: trypsin inhibitor: du's phosphate buffer: 1N NaOH 600:600:20: 150;

the weight volume ratio of each component in the low egg raw solution is as follows in mg/mg/mL/muL: bovine serum albumin: trypsin inhibitor: du's phosphate buffer: 1N NaOH 300:300:20: 100.

In some embodiments of the invention, the culturing in step E employs a medium of RGCs that consists of:

in some embodiments of the invention, the DMEM-SATO basal medium consists of:

in some embodiments of the present invention, the centrifugation in step D is centrifugation at 900rpm for 5min at 18-25 ℃.

The invention improves the related method for separating and purifying the RGCs, and multiple experiments prove that the number of the RGCs obtained by averaging a single retinal tissue is obviously higher than that of the original two-step immunodomination method, and the purity of the separated RGCs is also obviously higher than that of the two-step immunodomination method. Different batches of RGCs were immunofluorescent stained using the specific marker antibody β -tubulin III, BRN 3A. The results show that the purity of the RGCs marked by the two antibodies has no statistical difference, and further verify that the improved two-step immune disrotatory method can obtain primary RGCs with relatively stable purity and yield. The research lays a cytological foundation for large-scale deep research on the mechanism of vision deterioration caused by RGCs necrosis and apoptosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the preparation of positive and negative petri dishes;

FIG. 2 shows a coated cell culture plate;

FIG. 3 shows a detached retina;

FIG. 4 shows the preparation of a primary retinal cell suspension;

FIG. 5 illustrates the preparation of a low-egg stock solution, a high-egg stock solution;

FIG. 6 shows retinal cells bound to specific antibodies;

FIG. 7 shows the screening of cells using negative and positive culture dishes;

FIG. 8 shows RGC medium resuspending cells, plated after cell counting;

FIG. 9 shows isolation of primary SD rat RGCs using a "modified two-step immunodeplication method"; among them, FIG. 9(A) shows that the green fluorescent cells are RGCs labeled with β -tubulin III; red fluorescence is RGCs labeled with antibodies specific for BRN 3A; blue is the nucleus marked with DAPI. Scale bar: 20 μm; FIG. 9(B) shows that the cell purities of β -tubulin III and BRN3A, which are labeled with specificity for RGCs, are not statistically different from each other, and the results show that the cell purities obtained by the "modified two-step immuno-dishing method" are relatively stable, and the data are expressed as means. + -. standard error; 90.06 +/-0.1696 percent of beta-tubulin III; BRN3A:88.51 ± 0.5859% (n ═ 3; ns: P ═ 0.0632> 0.05);

FIG. 10 shows the average yields of RGCs from primary SD rats isolated using different immunodeplication methods; data are expressed as mean ± sem. 18890 + -484.4 cells/retina; an improved two-step immunoplate method comprising 203000 + -6173 cells/retina (n: 3;. P < 0.0001);

FIG. 11 shows comparison of purity of primary SD rat RGCs isolated by different immunoseparations; FIG. 11(A) shows that the green fluorescence is RGCs labeled with β -tubulin III, and the blue fluorescence is DAPI-stained nuclei at a scale bar of 20 μm; FIG. 11(B) data are expressed as mean. + -. standard error; the purity of RGCs by the two-step immune disrotatory method is 72.29 +/-1.025%; an improved two-step immunopterification method comprising 90.06 + -0.1696% (n ═ 3;. P < 0.0001).

Detailed Description

The invention discloses a preparation method of retinal ganglion cells, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

RGCs are neurons located in the innermost ganglion cell layer of the retina and are composed of multipolar ganglion cells. The dendrites are primarily associated with bipolar cells, but can also be associated with podocytes laterally. RGCs converge on elongated axons in the brain to form the optic nerve, which is an important channel for conducting visual signals. They play a crucial role in transmitting the visual information of photoreceptors to the brain, accelerating the necrosis and apoptosis of RGCs during the damage of optic nerve caused by glaucoma, diabetic retinopathy and trauma, resulting in visual impairment to visual loss. Therefore, the research on the RGCs is deepened, the mechanism of injury and apoptosis is discussed, the understanding on the pathological process of glaucoma and diabetes is further deepened, the visual loss is delayed, and the prognosis of patients is improved. Therefore, studies related to RGCs are a bridge between basic pathology studies and clinical treatment of patients. However, RGCs, a central neuron, are very sensitive to the external environment and are susceptible to disintegration and apoptosis in vitro. Moreover, the RGCs obtained by the original two-step immunodomination method are few in quantity and limited in purity, cannot meet a large number of cell experiments, are not beneficial to the research on visual deterioration caused by eye diseases such as glaucoma and diabetes, and cause great obstacles to the basic research on related diseases of the RGCs, so that the search for a more stable and effective method for purifying and culturing primary RGCs is more urgent.

In our research, innovations are made on the principle of using the coated antibody, and the method for extracting RGCs by the two-step immune disklization method is improved, so that the whole process is shorter in time, more cells are obtained, and the cell purity is higher. The purity (90.06 + -0.1696%) of RGCs purified by the improved two-step immune disrotatory method is obviously higher than that of RGCs purified by the two-step immune method (72.29 + -1.025%). The original "two-step immunoplate" procedure, in the positive selection procedure, cell suspension was incubated for 45 minutes. Through a large amount of research, the following results are found: during the 45 minute positive selection, endothelial cells, macrophages and microglia settle to the bottom of the negative selection dish during this time period due to gravity as the culture time increases, and the presence of contaminating cells increases due to the attractive interaction between cells, resulting in an increase in the number of non-RGCs, resulting in a substantial decrease in purity. While in our "modified two-step immunoplate" we used 10 minutes incubation time depending on the principle used, in the positive panning process, the time taken for the RGCs to settle to the bottom of the dish is shorter than the time taken for other non-RGCs due to the binding of the antibody to the cell surface specific antigen because the bottom of the dish is coated with CD90 antibody. Therefore, the improved two-step immune disrotatory method is to separate and purify the RGCs by utilizing the time difference of sedimentation between the RGCs and non-RGCs, shortens the whole purification process and can obtain the RGCs with high purity and high yield. The cell yield (203000 +/-6173/retina) of the RGCs of the improved two-step immune disketting method is far higher than that of the original two-step immune disketting method (18890 +/-484.4/retina). Moreover, when separating the RGCs settled to the bottom of the positive screening culture dish, a separation method of digesting by digestive enzyme after rinsing by a large amount of D-PBS in the original 'two-step immunization disk method' is not used, and the method can cause a large amount of RGCs to be lost in the rinsing process of the D-PBS, so that the yield of cells is greatly reduced, the activity of retinal ganglion cells is greatly reduced after the enzymatic digestion, and partial cells directly die. The improved two-step immune disklization method utilizes the characteristic that the combination of CD90 and RGCs surface antigen is not very tight, and uses a physical method of gentle blowing and beating by D-PBS to separate the antibody coated at the bottom of the culture dish from the antigen of the cell, thus for the RGCs with weak in vitro vitality, the activity of the cell can be maintained to the maximum extent, the influence of external factors is reduced, and the separated RGCs can be close to the original vitality state to the maximum extent.

In conclusion, the purity of the RGCs obtained by the improved two-step immune disketting method is about 90 percent and is obviously higher than that of the RGCs obtained by the original two-step immune disketting method by 72.29 +/-1.025 percent; and the yield of the obtained cells is about 10 times that of the original two-step immune discriminant method (18890 +/-484.4/retina) by the improved two-step immune discriminant method (203000 +/-6173/retina). Therefore, the improved two-step immunization discriminant method is obviously superior to the original two-step immunization discriminant method, so that the acquisition of the primary SD rat RGCs with high purity and high yield is possible, and a solid cytology foundation is laid for the research of vision reduction mechanisms caused by diseases such as glaucoma, diabetic retinopathy and the like.

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

Animals: primary retinal ganglion cells were extracted from young SD rats born for 48-72h and purchased from the center of Zhengzhou university laboratory animals, and all the animals were sacrificed by cervical dislocation.

The formulation of reagents used for RGCs cell culture is shown in the following table:

TABLE 1 RGCs Medium composition

Reagent	Adding amount of
		DMEM-SATO basal Medium (Table 2)	20mL
Maohuosu (4.2mg/mL) (Table 3)	20μL
		Brain-derived neurotrophic factor (50 mug/mL)	20μL
Ciliary neurotrophic factor (10 ug/mL)	20μL

TABLE 2 DMEM-SATO basal Medium composition

Mixing the above reagents, and sterilizing with 0.22 μm filter

TABLE 3 forskolin preparation table (4.2mg/mL)

Reagent	Adding amount of
		Forskolin (Sigma-Aldrich F6886)	10mg
Dimethyl sulfoxide (Solambio D8371)	2.4mL

Mixing, filtering with 0.22- μm polytetrafluoroethylene filter, sterilizing, packaging 100 μ L, and storing at-20 deg.C.

TABLE 4 insulin preparation Table (0.5 mg/mL; Absin abs9169-25mg)

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

Mix well on ice and filter to remove bacteria through a 0.22 μm filter. Stored at 4 ℃.

TABLE 5 thyroxine (T3) preparation Table (8mg/mL)

Reagent	Adding amount of
		Thyroxine sodium salt (T3; Sigma-Aldrich T6397)	1.6mg
0.1N NaOH	200μL

Prepare 4 μ L/mL thyroxine (T3) stock: mu. L T3 solution (8mg/mL) was added to 10mL phosphate buffered saline (D-PBS; Gibco 14287). Sterilized by filtration through a 0.22 micron filter, aliquoted into 500 μ L aliquots and stored at-20 ℃.

TABLE 6N-acetyl-L-cysteine Table of formulations (5mg/mL)

Mixing, filtering with 0.22 μm filter to remove bacteria, packaging into 100 μ L aliquots, and storing at-20 deg.C.

TABLE 7 SATO (the abbreviation is hereinafter abbreviated as "reagent English") additive preparation Table (100 ×)

Reagent	Adding amount of
		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

20ml of DMEM was added to the above reagent, gently stirred, sterilized with a 0.22 micron filter, aliquoted into 500 microliter aliquots and stored at-20 ℃.

TABLE 8 preparation of sodium selenite stock solution

Reagent	Adding amount of
		Sodium selenite (Sigma-Aldrich S5261)	1mg
DMEM(Gibco 11960-044)	2.5mL
		1N NaOH	2.5μL

Mixing lightly and keeping it for use.

TABLE 9 Progesterone stock solution preparation table

Reagent	Adding amount of
		Progesterone (Sigma-Aldrich P8783)	1mg
75% ethanol	40μL

Mixing lightly and keeping it for use.

Reagent formulation table used in isolation and purification:

TABLE 100.2% bovine serum albumin solution (0.2% BSA) preparation Table

Reagent	Adding amount of
		Bovine Serum Albumin (BSA) (Sigma-Aldrich A4161)	40mg
Du's phosphate bufferWashing liquid (D-PBS) (Hyclone SH30264.01)	20mL
		1N NaOH	5μL

Mixing well, sterilizing with 0.22 μm filter, and storing at-20 deg.C.

TABLE 11 DNase preparation Table

Reagent	Adding amount of
		DNase (Worthington LS002007)	3mg
Balanced Salt Solution (EBSS) (Solarbio H2040)	2mL

Formulated on ice, sterilized with a 0.22 micron filter and stored at-20 ℃.

TABLE 12 table of "high egg stock solution" preparation

Reagent	Adding amount of
		Bovine serum albuminProtein (BSA) (Sigma-Aldrich A4161)	600mg
Trypsin inhibitor (Worthington LS003086)	600mg
		Du's phosphate buffer (D-PBS) (Invitrogen 14287-080)	20mL
1N NaOH	150μL

Mixing well, filtering with 0.22 μm filter, and sterilizing. Storage at-20 ℃.

TABLE 13 formulation Table of "Low egg stock solution

Reagent	Adding amount of
		Bovine Serum Albumin (BSA) (Sigma-Aldrich A4161)	300mg
Trypsin inhibitor (Worthington LS003086)	300mg
		Du's phosphate buffer (D-PBS) (Invitrogen 14287-080)	20mL
1N NaOH	100μL

Mixing well, filtering with 0.22 μm filter, and sterilizing. Storage at-20 ℃.

TABLE 14 formulation Table for the discotic buffer

TABLE 15 Tris (hydroxymethyl) aminomethane HCl buffer (Tris-HCl) (50mM, pH 9.5)

Reagent	Adding amount of
		Tris (hydroxymethyl) aminomethane (Tris)	12.1g
Distilled water	200ml

Mixing, adjusting pH to 9.5 with dilute hydrochloric acid, and sterilizing at high temperature and high pressure.

The invention is further illustrated by the following examples:

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

The first day:

1. preparation of Positive and negative selection plates (see FIG. 1)

1.1 two 50ml tubes were prepared and labeled "negative selection", and 40 ml of 50mM Tris-HCl (Tris-HCl buffer) (pH 9.5) and 120. mu.L goat anti-rabbit immunoglobulin (H + L) (Jackson 111-005-003) were added to each tube and gently mixed for further use.

1.2 preparing four negative selection culture dishes marked A1, A2, B1 and B2;

adding 20mL of mixed solution of the antibody and Tris-HCl into each 15cm culture dish, gently mixing uniformly, sealing by using a sterilization sealing film, and placing in a 4-DEG refrigerator overnight for later use.

1.3 one 50ml centrifuge tube was labeled "positive selection", 20ml of 50mM Tris-HCl (pH 9.5) and 60. mu.l of goat anti-mouse immunoglobulin IgM (H + L) (Jackson 115-005-044) were added and gently mixed.

1.4 prepare 2 10cm sterile petri dishes, labeled A and B. Add 10ml of mixed "Positive selection" antibody and Tris-HCl mix to each dish, mix gently, seal with sterile sealing film, and refrigerate overnight at 4 ℃ for use.

2. Coated cell culture plate (FIG. 2)

2.1 the number of 24 well plates was selected as required for the grouping and 500. mu.L of polylysine solution (Sigma-Aldrich P4707-50ML) was added to each 24 well plate to cover the bottom of the plate uniformly. And standing at room temperature for at least 2 hours to fully coat.

2.2 the laminin (Gibco 23017-015) was diluted to a concentration of 10. mu.g/ml using a neural basal medium (Gibco 21103-049) and mixed until ready for use. The air-borne polylysine was recovered and then diluted laminin was added, 500. mu.L per well. Then placed in a 37 ℃ incubator overnight.

The next day:

1. petri dishes for positive screening using primary coating

1.1A solution of 13.5ml of D-PBS (HyClone SH30264.01) and 1.5ml of 0.2% BSA (SigmaAldrich A1933-25G) was taken, and then 100. mu.L of mouse anti-rat CD90 antibody (Abd serotec MCA04G) was added thereto and gently blown and mixed for use.

1.2 2 the 2 prepared 10cm "positive selection" dishes from the first day were rinsed 3 times with D-PBS. 7.5mL of mouse anti-rat CD90 antibody solution was added to each dish and incubated for 2 hours at room temperature (18 ℃ -25 ℃).

2. Separating retinas

2.1 retina isolation procedure (FIG. 3)

2.1.1 sacrifice of newborn SD rats 48-72h after birth by cervical dislocation, placing in 75% alcohol for 3 minutes, then rapidly removing their eyes, placing in a pre-contained cold D-PBS culture dish. Retinal tissue in the eyeball was isolated using a dissecting microscope.

First, the eyeball was fixated with a pair of tweezers, a small hole was cut with a pair of microscissors, and then cutting along the corneal scleral rim was started. The lens and cornea of the anterior portion of the eye are removed. Secondly, the tail end of the optic nerve is fixed right in front of the eyeball by a pair of micro-tweezers, and the eyeball is slightly pressed by another pair of micro-tweezers in the opposite direction, so that the retina is floated out of the sclera. Finally, the vascular membrane attached to the retina was gently pulled out and removed, and the retina was transferred to a 1mL EP tube filled with cold D-PBS for storage.

3. Preparation of primary retinal cell suspension (FIG. 4)

3.1 three 15mL centrifuge tubes were prepared as described below

3.1.1A centrifuge tube was filled with 5ml of D-PBS and 70. mu.L of papain (Worthington Biochemical LS003126) labeled "papain tube". After mixing, the mixture is bathed for 5 minutes at 37 ℃.

3.1.2 Add 9mL of D-PBS to the 15mL centrifuge tube, labeled "Low egg stock solution tube", and add 5mL of D-PBS to the other centrifuge tube, labeled "high egg stock solution tube", for use.

3.2 weigh 1 mg of L-cysteine and add it to the "papain tube" and adjust the pH by adding 5. mu.L of 1N NaOH.

3.3 in a "papain tube", 190. mu.L DNase (Worthington LS002007) was added and mixed well, followed by filter sterilization using a 0.22 μm (Millipore SLGV033RS) filter.

3.4 the prepared retinal tissue was transferred to a filter sterilized papain tube using a 1mL pipette gun and heated in a 37 ℃ water bath for 20 minutes to digest the tissue.

3.5 preparation of "Low egg stock" and "high egg stock" (FIG. 5)

Adding 1mL of the low egg stock solution and 2 muL of 1N NaOH into a centrifuge tube marked with the low egg stock solution to be mixed uniformly; 1mL of the high egg stock solution and 2. mu.L of 1N NaOH are added into a centrifuge tube marked with the high egg stock solution and mixed uniformly for later use.

3.620 minutes later, the supernatant in the "papain tube" was aspirated and 4ml of "low egg stock" was added dropwise to the tube. The mixture was allowed to stand for 3 minutes to terminate the papain digestion.

4 retinal cells binding to specific antibodies (FIG. 6)

4.1 mu.L rabbit anti-rat macrophage polyclonal antibody (Cedarlane CLAD51240) was added and mixed well with the remaining 6ml "low egg stock" for use.

4.2 gently blow and beat the 'low egg stock solution', and 2 ml of the mixed solution of the 'low egg stock solution' and rabbit anti-rat macrophage antibody is added into the digested retinal tissue. The retina was abraded 8-10 times with a 3ml disposable pipette and the solution was allowed to stand for 1 minute.

After 4.31 minutes, the supernatant was transferred to a new 15ml tube. The original tube was again added with 1ml of "low egg stock solution + anti-macrophage antibody" (90. mu.L of rabbit anti-rat macrophage polyclonal antibody (Cedarlane CLAD51240) and mixed with the remaining 6ml of "low egg stock solution" to prepare a "low egg stock solution + anti-macrophage antibody mixed solution"), and the retina was ground 8 to 10 times and allowed to stand for 1 minute. The same procedure was repeated until the "low egg stock solution + anti-macrophage antibody" mixed solution was completely used up.

4.4 the retinal cell suspension obtained after grinding is incubated at room temperature for a further 10 minutes to allow the rabbit anti-rat macrophage antibody to bind to the specific cellular epitope.

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

5. Screening of retinal ganglion cells was performed using coated negative and positive culture dishes (FIG. 7)

5.1 preparation of buffer:

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

5.2 the single cell suspension after resuspension was filtered using a 40 μm nylon mesh to remove undigested tissue.

5.3 transfer the filtered cell suspension to two 15cm large negative selection dishes labeled A1 and B1. The mixture was allowed to stand at room temperature for 25 minutes.

5.425 min, the cells were transferred to two additional 15cm negative selection plates labeled A2 and B2, respectively, and left to stand at room temperature for 30min to remove macrophages, microglia and endothelial cells from the cell suspension by the above negative selection.

5.5 discard two 10cm positive screening culture dishes of mouse anti-rat CD90 antibody solution, D-PBS rinse 3 times, through negative screening cell suspension transfer to mark A and B positive screening culture dishes, the room temperature 10 minutes.

5.610 minutes later, the supernatant was removed from a 10cm positive screening dish using a disposable 3mL pipette. The plates were examined under a microscope to ensure that only cells remained. Then, a 10cm petri dish was washed with an appropriate amount of D-PBS, and cells adsorbed on the bottom of the petri dish were washed away. The dish was examined under a microscope to ensure that no cells were attached to the bottom of the dish.

5.7 transfer the cell solution in two 10cm petri dishes to two 15ml centrifuge tubes and labeled A, B centrifuge tubes. Then, the mixture was centrifuged at 900rpm at room temperature (18 to 25 ℃) for 5 minutes.

6. Resuspend cells in RGCs media and count using cell counting plates (FIG. 8)

6.1 resuspension of RGCs, cell density was adjusted to 10⁵/mL。

6.2 washing the coated 24-well plate 2-3 times with D-PBS, and mixing the RGCs at 10⁵The density of/mL was plated in 24-well plates. 5% CO at 37 deg.C₂And 95% airCulturing in an incubator.

Further purification of RGCs

7.1 to better reflect the characteristics of the cells, and to meet the needs of the experiment, further purification of RGCs is required.

7.2 days later, the medium is replaced with a new one (as shown in Table 1), the RGCs which are dead and in bad state are removed, and the cell purity can reach about 90% after the treatment (detection method: the cell purity detection adopts two specific markers of the RGCs, the RGCs extracted from different batches are randomly subjected to fluorescent staining, the number of the cells which are positively stained by the specific markers is divided by the number of the cells stained by DAPI and multiplied by 100%, and the cell purity is taken as a measurement standard of the cell purity, the detection condition is 18 to 25 ℃ at room temperature, and the requirements of subsequent experiments are met.

Example 2 immunofluorescent staining

After 24 hours of plating, the obtained cells were subjected to immunofluorescence staining using a specific marker, and the purity of the cells was determined. After 24 hours of plating, the RGCs were discarded from the cell culture medium, rinsed 3 times with PBS (Solarbio P1020-500mL), and fixed in an immunostaining solution (Beyotime P0098-100mL) at room temperature for 15 min. Immunostaining washes (Beyotime P0106) were washed 3 times for 10min each. After rinsing, blocking was performed for 1h at room temperature using immunostaining blocking solution (Beyotime P0102). This was followed by overnight incubation at 4 degrees with either an anti- β -tubulin III antibody (1:500, Abcam ab7751) or an anti-BRN 3A antibody (1:500, bs-3669R). Wash 3 times for 5min using immunostaining washes (Beyotime P0106). Then incubated with the corresponding secondary antibody (donkey anti-mouse IgG H & L AlexaFluor488, 1:1000 dilution, Abcam ab 150105; donkey anti-rabbit IgG H & L AlexaFluor 647,1:1000 dilution, Abcam, ab150075) for 2 hours at room temperature in the absence of light. Then, the cells were washed 3 times for 5 minutes each using an immunostaining wash (Beyotime P0106). DAPI (Beyotime C1005) was counterstained for 5 minutes of nuclei at room temperature, followed by rinsing with immunostaining washes 3 times for 5 minutes each. After the completion of the rinsing, an appropriate amount of an anti-immunofluorescence quencher (Abcam ab104135) was added dropwise. Using OLYMPUS fluorescence microscope software to excite AlexaFluor and DAPI at 488nm, 568nm and 358nm respectively, randomly selecting 1 visual field under a 400X fluorescence microscope to take a picture, repeating the steps for 3 times in each group, using Image J software to analyze the obtained pictures to calculate the purity and yield of cells, and comparing the purity and yield with the original immune separation method.

Examples of effects

1.1 extraction of purity of Primary SD rat retinal ganglion cells Using "improved two-step Immunodification method

Different batches of primary RGCs obtained were randomly labeled with Different specific markers (Differencent makers) β -tubulin III or BRN3A (FIG. 9), indicating that: the cellular Purity of β -tubulin III labeled RGCs (Purity of RGCs) was: 90.06 +/-0.1696%; the cell purity of BRN 3A-labeled RGCs was 88.51. + -. 0.5859%, with no statistical difference between the two. This result demonstrates that the Improved two-step immunopanning method can obtain RGCs with higher cell purity and stability.

TABLE 16 comparison of purity of extracted cells of different batches by immunofluorescence staining of different specific markers of RGCs

P ═ 0.0632>0.05, indicating that primary RGCs of higher purity and stable percentage can be obtained using the "modified two-step immunoplate method" using random detection with different specific markers (experiments were repeated three times).

1.2 yield of Primary SD rat retinal ganglion cells obtained by different immuno-isolation purification methods

The cell yields (Yield of purification) obtained by the original "Two-step immunopanning method" (Two-step immunopanning method) and the "Improved Two-step immunopanning method" (Improved Two-step immunopanning method) were counted and the Yield of primary SD rat retinal ganglion cells obtained by the Two different methods was quantitatively analyzed. There was a large difference in the RGCs production by the two methods. The average yield of the "two-step immunization" was 18890. + -. 484.4 cells/retina and the yield of the "modified two-step immunization" was approximately 203000. + -. 6173 cells/retina. The yield of the improved two-step immunization disk method is obviously higher than that of the original two-step immunization disk method (figure 10).

TABLE 17 comparison of yields of primary RGCs cells obtained by two different immuno-isolation purification methods

P is less than 0.0001, the yield of primary RGCs obtained by two different immune separation and purification methods is obviously different, and the improved two-step immune dishing method is far higher than the two-step immune dishing method (the experiment is repeated three times).

1.3 purity of primary SD rat retinal ganglion cells obtained by different immuno-separation methods.

From the results of 1.1, it was found that β -tubulin III or BRN3A, which are specific markers for RGCs, had no statistical difference in determining the purity of the labeled RGCs. Therefore, we used only beta-tubulin III for labeling, and compared the cell purity obtained by different immuno-dishing methods. The purity of RGCs isolated by the "two-step immunopischemization method" was 72.29 + -1.025%, which was significantly lower than that of "modified two-step immunopischemization method" 90.06 + -0.1696% (FIG. 11), and there were statistical differences (P < 0.0001).

TABLE 18 comparison of the purity of RGCs obtained by two different immunological separation and purification methods

P is less than 0.0001, the purity of primary RGCs obtained by two different immune separation and purification methods is obviously different, and the cell purity of the improved two-step immune dishing method is far higher than that of the two-step immune dishing method (the experiment is repeated three times).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (1)

1. The preparation method of the retinal ganglion cells is characterized by comprising the following steps of:

step A, obtaining a retinal cell suspension;

step B, preparing and obtaining a negative screening vector and a positive screening vector; the negative screening vector is coated with a goat anti-rabbit IgG (H + L) secondary antibody, and the positive screening vector is coated with a goat anti-mouse IgM (H + L) secondary antibody;

step C, coating the positive screening carrier with primary antibody;

step D, mixing the rabbit anti-rat macrophage polyclonal antibody with the retinal cell suspension prepared in the step A, incubating and centrifuging to obtain retinal cells;

step E, taking the retinal cells prepared in the step D for re-suspension, screening the negative screening carrier prepared in the step B, collecting cell suspension, screening the positive screening carrier prepared in the step B, removing supernatant, collecting cells, culturing and purifying;

wherein, the sequence of the step A and the step B is not sequential;

e, collecting cells, separating retinal cells in a blowing mode without using digestive enzymes;

e, the incubation time for screening the positive screening carrier prepared in the step B is 10 min;

the culture in step E adopts RGCs culture medium, which consists of the following components:

the DMEM-SATO basal medium consists of the following components:

；

step A, obtaining a retinal cell suspension by using digestive enzyme; the digestive enzyme comprises papaya digestive enzyme;

in the step D, the incubation is carried out for 10min at the temperature of 18-25 ℃;

the primary antibody in the step C is a mouse anti-rat CD90 antibody;

and D, centrifuging at 900rpm for 5min at 18-25 ℃.

Images