KR102196069B1 - Method for Isolating of Retinal Ganglion Cell and Uses of the Cells Thereby Isolated - Google Patents

Abstract

The present invention relates to a method for isolating retinal ganglion cells and to the use of the cells separated therefrom. According to an embodiment of the present invention, retinal ganglion cells can be effectively separated or concentrated, and the intracellular environment or cell integrity Since primary retinal ganglion cells can be isolated and cultured without affecting cellular integrity, they can be usefully used in subsequent studies related to glaucoma or optic neuropathy.

Description

Method for Isolating of Retinal Ganglion Cell and Uses of the Cells Thereby Isolated}

It relates to a method for isolating retinal ganglion cells and to use of the cells isolated thereby.

The retina has a structure made up of several layers. Among them, the neuronal layer is composed of three layers of neurons by photoreceptor cells, bipolar cells, and retinal ganglion cells (RGC). Photoreceptor cells are neurons that respond to light, bipolar cells process nerve signals, and retinal ganglion cells form optic nerve axons to collect nerve signals in the form of action potentials.

The loss of retinal ganglion cells and their axons due to retinal degenerative diseases leads to visual impairment and poor quality of life. In particular, glaucoma is one of the leading causes of blindness worldwide, and is a progressive optic neuropathy that indicates an optic nerve papillary injury and a characteristic visual field defect. As a characteristic aspect of glaucoma, selective destruction of retinal ganglion cells and damage to the optic nerve are known, but the exact cause or mechanism of glaucoma is not yet known. Since elevated intraocular pressure as a risk factor for rust catastrophe is the best known, glaucoma research over the past decades has focused primarily on lowering intraocular pressure. However, normal-tension glaucoma is a progression of glaucoma while maintaining a low range of intraocular pressure from the time of onset, and its incidence has been reported to be high in Asia including Korea. Therefore, in recent years, instead of an indirect method of lowering intraocular pressure, attempts have been made to find a neuroprotection mechanism that can more directly prevent the death of retinal ganglion cells.

Primary cells are cells that have been primary cultured directly from animal tissues or organs, and refer to normal cells directly obtained from living organisms. Primary cultured cells are being developed as cell therapy products, and because they have the advantage of being similar to the actual reactions of living organisms, they are used in the production of biological drugs. Primary retinal ganglion cells can be usefully used in the study of diseases caused by degeneration and damage of the optic nerve.

Therefore, there is a need for a method capable of separating primary retinal ganglion cells with high yield and stably enriching cells.

Republic of Korea Public Publication No. 10-2017-0077117 (2015.09.05)

One embodiment provides a method of separating retinal ganglion cells.

Another embodiment provides a method of screening a retinal ganglion cell death inhibitory substance or a concentration substance using the retinal ganglion cells isolated by the above method.

In one aspect, obtaining cells from retinal tissue; Inoculating and culturing the obtained cells in a container coated with a cell adhesion protein; Shaking the container after incubation; And it provides a method of separating retinal ganglion cells (retinal ganglion cells) comprising the step of collecting the detached cells from the container.

"Retinal ganglion cells (RGC)" are projection output nerve cells that exist in the retina of vertebrates, and axons of these cells gather to form the optic nerve. Retinal ganglion cells are neurons, that is, neurons, which are the building blocks of the nervous system, and have multiple dendrites and long fibrous axons. The dendrites form a synapse by bonding with other cells, and they control the electrical activity of nerve cells to transmit electrical impulses flowing into the cell body. Axons have many branches at the ends, and there are granules or vesicles, and these contain neurotransmitters secreted by nerve stimulation.

According to one embodiment, the step of obtaining cells from the retinal tissue may include preparing the retinal tissue as an aqueous cell solution; Filtering the aqueous cell solution with a cell filter; And collecting cells from the filtered aqueous cell solution.

The term "cell aqueous solution" refers to a solution in which cells constituting a tissue are separated. According to one embodiment, the step of preparing the retinal tissue as an aqueous cell solution may include preparing an aqueous cell solution from the retinal tissue. To prepare the aqueous cell solution, the retinal tissue may be suspended in a buffer solution and the aqueous cell solution may be prepared using a chemical method and/or a physical method.

The chemical method may be to use a buffer solution containing an enzyme. The buffer solution may be used without particular limitation as long as it is used in the treatment of animal cells in the art. For example, phosphate buffered saline (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid) buffer solution, Bicine ( N,N-Bis(2-hydroxyethyl)glycine) buffer solution, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) buffer solution , PIPES (piperazine-N,N'-bis (2-ethanesulfonic acid) buffer solution, cacodylate buffer solution, or MES (2-(N-morpholino)ethanesulfonic acid) buffer solution, etc., but are limited thereto. It is not.

Enzymes break the junction between cells that make up the tissue, thereby reducing the size of the tissue and allowing the cell layer to be separated from the tissue. The enzyme may be one or more selected from the group consisting of collagenase type I, collagenase type II, collagenase type IV, trypsin, EDTA, Dispase, and Accutase.

The physical method may be to separate the cells from the tissue by shaking or pipetting the aqueous cell solution. The shaking or pipetting may be performed in a range in which the plasma membrane of the animal cell is not damaged.

According to one embodiment, the step of obtaining cells from the retinal tissue may include filtering the aqueous cell solution with a cell filter. The pore size of the cell strainer may be 35 to 45 μm, 39 to 45 μm, 35 to 41 μm, 39 to 41 μm, or 40 μm. For example, when filtering through a cell filter having a pore size of 40 μm, retinal ganglion cells smaller than 40 μm may pass through the filter.

According to one embodiment, the step of preparing the retinal tissue as an aqueous cell solution may include collecting cells from the filtered aqueous cell solution. The collection may be to resuspend the precipitate obtained by centrifuging the aqueous cell solution passing through the filter at 1500 rpm for 3 minutes.

According to one embodiment, the method of separating retinal ganglion cells may include inoculating and culturing the obtained cells in a container coated with a cell adhesion protein.

The cultivation may be performed according to an appropriate medium and culture conditions known in the art. Media refers to a medium capable of supporting cell growth and survival in vitro. According to one embodiment of the present invention, the medium may be a complete medium. According to one embodiment, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal Essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE (Bovine Pituitary Extract)), Keratinocyte-SFM (with BPE), KnockOut D-MEM, AmnioMAX-II It may be selected from the group consisting of Complete Medium and AmnioMAX-C100 Complete Medium, but is not limited thereto.

The inoculation can inoculate an appropriate number of cells according to the type and size of the container. For example, 2.5×10 ⁷ cells may be inoculated based on a T-70 flask, and cultured for 5 days at 37±1° C. and 5% CO ₂ . The container may be a plate, a T-flask, a culture dish, a microwell plate (6, 12, 24, or 96 well), a tube, or a roller bottle.

The cell adhesion protein is one capable of inducing adhesion or adhesion of cells and may be, for example, collagen, gelatin, fibronectin, laminin, or poly-D-lysine. According to one embodiment, the cell adhesion protein may be laminin. When the container is coated with laminin, the retinal ganglion cells are more easily attached and separated by shaking than when coated with poly-D-lysine or collagen, so that the retinal ganglion cells can be separated more easily.

According to one embodiment, the method of separating retinal ganglion cells may include shaking the container after culture. In the shaking step, according to a difference in binding affinity to the cell adhesion protein, cells attached to the container and separated cells may be separated. For example, astrocytes or Muller cells are attached to a container coated with a cell adhesion protein even after shaking, and retinal ganglion cells are detached, so retinal ganglion cells can be easily separated. For example, when laminin is coated as a cell adhesion protein, retinal ganglion cells can be effectively separated.

According to one embodiment, the shaking is 100 to 400rpm, 150 to 400rpm, 200 to 400rpm, 250 to 400rpm, 300 to 400rpm, 100 to 350rpm, 150 to 350rpm, 200 to 350rpm, 250 to 350rpm, 300 to 350rpm, 100 To 300 rpm, 150 to 300 rpm, 200 to 300 rpm, or 250 to 300 rpm. According to one embodiment, the shaking may be shaking for 120 to 240 minutes, 150 to 210 minutes, 170 to 190 minutes, or about 180 minutes. According to one embodiment, the shaking step may be performed using, for example, an orbital shaker or a plate shaker. According to one embodiment, the shaking may be performed for 2 to 3 hours or 3 hours at 100 to 300 rpm, 150 to 300 rpm, 200 to 300 rpm, or 250 to 300 rpm using an orbital shaker. When shaking at the rpm and time, the retinal ganglion cells can be efficiently released, and the ratio of retinal ganglion cells in the detached cells can be high.

According to one embodiment, the method of separating the retinal ganglion cells may include collecting the separated cells from the container. The step of collecting the cells released from the container may be performed by centrifuging a culture medium containing the cells released from the container to precipitate the cells, and inoculating the precipitated cells into a petri dish. In one embodiment, the centrifugation may be performed at 1300 to 1700 rpm, 1400 to 1600 rpm, or 1500 rpm, and the centrifugation time may be 2 minutes to 4 minutes, 2 minutes 30 seconds to 3 minutes 30 seconds, or 3 minutes. .

According to one embodiment, the step of obtaining cells from the retinal tissue may be using a retinal tissue isolated from a mammal, and in detail, using a retinal tissue isolated from a rat or mouse. have.

According to one embodiment, the age of the rat may be within 1 day after birth, within 2 days after birth, or within 3 days after birth. Rats of this age are small in size because differentiation or growth of retinal ganglion cells is not completed, and are easily separated by filtration, and the cells can survive for a relatively long period even after separation or enrichment. In addition, rats of this age do not contain or contain relatively few immune cells (e.g., macrophages or microglial cells) in the retinal tissue, so they can be efficient in the separation and enrichment of retinal ganglion cells. have. Since the macrophages or microglial cells are easily separated from the cell adhesion protein in the shaking step, it may be advantageous to separate and enrich the retinal ganglion cells to use a retinal tissue containing less. Referring to the CD4 staining pictures of Example 1 and FIG. 5 of the present application, immune cells (e.g., macrophages or microglial cells) are absent in the retina of a rat on day 1 to 3 or 2 after birth, or Since it contains relatively less than that of an adult, retinal ganglion cells can be efficiently separated or concentrated from the retinal tissue of the rat on the second day of life.

According to one embodiment, the method of separating the retinal ganglion cells comprises the step of re-inoculating the collected cells in a container coated with a cell adhesion protein after the step of collecting the detached cells and rearranging the cells. It may contain more.

The step of re-inoculating the collected cells in a container coated with a cell adhesion protein and culturing may be performed in an incubator under conditions of 37±1° C. and 5% CO ₂ . "Cell enrichment" refers to increasing the concentration or density of a specific cell among several mixed cells, and can be achieved by purification, filtration, separation, or culture conditions optimized for a specific cell. Cell concentration may increase the concentration of specific cells in the primary culture and at the same time do not change the characteristics of the cells. According to an embodiment of the present invention, the cultivation may be cultured for 3 days, 5 days, 7 days, or 10 days, and the morphological characteristics of the retinal ganglion cells during the cultivation period (for example, RGC is The nucleus (round nucleus), extended axons (stretched-out axon) or a number of dendrites (dendrite) can be maintained.

Cells isolated by the method according to an embodiment may immunochemically have a proportion of Tuj-1 positive and Nestin positive cells of 80% or more, or 85% or more, and a proportion of Nestin-positive and GFAP-negative cells of 80% Or more, or 85% or more. If the Tju-1 protein is positive, the presence of nerve cells can be confirmed, and if the Nestin protein is positive, the presence of neural stem cells can be confirmed. Therefore, it can be seen that the higher the amount of Tju-1 and Nestin protein, the higher the proportion of retinal ganglion cells among the cells present in the separated culture medium. GFAP protein is a protein mainly expressed in Astrocyte and Muller cells, not retinal ganglion cells. Therefore, it can be seen that the smaller the amount of GFAP protein, the higher the proportion of retinal ganglion cells among the cells present in the isolated culture medium.

Another aspect is the step of treating the retinal ganglion cells separated by the method of separating the retinal ganglion cells with a candidate material; Comparing retinal ganglion cells treated with the candidate substance and retinal ganglion cells not treated with the candidate substance; And when the candidate substance inhibits retinal ganglion cell death or enriches retinal ganglion cells, determining the candidate substance as a retinal ganglion cell death inhibitory substance or an enrichment substance, respectively, of retinal ganglion cells. It provides a method of screening for apoptosis inhibitors or concentrated substances.

The term "retinal ganglion cell death inhibitory substance" refers to a substance capable of reducing or inhibiting the degree of death of retinal ganglion cells in a death-inducing condition. The death inhibitory substance may be, for example, a compound, protein, peptide or nucleic acid, and is not particularly limited.

The term "retinal ganglion cell enrichment material" refers to a material capable of inducing or promoting enrichment of retinal ganglion cells. The enrichment has the same meaning as the above-described "cell enrichment", and means that the concentration or density of retinal ganglion cells is increased. The increase in the concentration or density may be due to an increase in the number of cells, or may be an increase in a relative cell number ratio. According to one embodiment, the concentrated material may be, for example, a compound, protein, peptide, or nucleic acid, and is not particularly limited.

According to one embodiment, the death inhibitory substance or the concentrated substance may be a therapeutic agent for glaucoma or optic neuropathy.

The term "glaucoma" refers to a disease having damage to the optic nerve accompanied by loss of retinal ganglion cells. The term "optic neuropathy" refers to a disease in which retinal ganglion cells or axons of retinal ganglion cells are gradually lost, resulting in visual disturbance. The optic neuropathy may be optic neuritis, ischemic optic neuropathy, addiction-nutritional optic neuropathy, hereditary optic neuropathy, or optic nerve atrophy. Since glaucoma and optic neuropathy are diseases accompanied by loss of retinal ganglion cells, a substance capable of inhibiting or enriching the death of retinal ganglion cells can be used as a therapeutic agent for glaucoma or optic neuropathy.

The method of separating retinal ganglion cells according to an embodiment can effectively separate retinal ganglion cells.

The method of separating the retinal ganglion cells according to an embodiment can effectively enrich the retinal ganglion cells.

In the method of separating retinal ganglion cells according to an embodiment, intracellular properties or cellular integrity of the retinal ganglion cells may be maintained.

The method of separating retinal ganglion cells according to an embodiment can efficiently separate and concentrate primary retinal ganglion cells, so it can be usefully used in studies related to the onset of diseases related to loss of retinal ganglion cells or functional decline or treatment .

1 and 2 show the results of labeling the retinal tissues and thymus cells of P2, P7, and P14 rats with Thy-1 (green) through immunohistochemistry experiments. As a positive control, rat thymocytes were labeled with anti-Thy-1 antibody, and nuclei were counterstained with DAPI. GCL is the ganglion cell layer, IPL is the inner plexiform layer, NBL is the neuroblast layer, INL is the inner nuclear layer, and OPL is the outer plexiform layer. ), ONL represents the outer nuclear layer, RPE represents the Retinal Pigment Epithelial Cell, and IS/OS represents the photoreceptor inner segment/outer segment. Do). In FIG. 1, Merge is a diagram showing a result of staining with DAPI and a diagram showing the result of labeling with Thy-1. 1 and 2, the bar is 100 μm.
3 and 4 show the results of labeling the retinal tissues of P2, P7, and P14 rats with Nestin, Tuj-1, and GFAP. In FIG. 3, nestin, Tuj-1 and GFAP appear as green signals, and in FIG. 4, nestin appears as green and Tuj-1 as red signals, and the nuclei were counterstained with DAPI. 3 and 4, the bar is 100 µm. In FIG. 4, Merge is a diagram showing the result of staining with DAPI, and a diagram showing the result of labeling with Nestin and Tuj-1.
5 shows the expression of CD4 (green) in the retina and lymph nodes of rats. 'CD40' means CD4. In Figure 5, the bar is 100㎛. In Fig. 5, Merge is a merged view showing the result of staining with DAPI and the result of labeling with CD4.
6 shows a process of separating retinal ganglion cells (RGC) according to an embodiment.
7A and B show the results of observation using a phase contrast microscope before and after separating the retinal ganglion cells (RGC) in the agitation step in the method of separating retinal ganglion cells according to an embodiment. . Specifically, FIG. 7A shows cells in the pre-shaking stage, and FIG. 7B shows cells cultured for 7 days after shaking.
8A and 8B show the results of immunohistochemical staining of retinal ganglion cells before and after the shaking step. Specifically, FIG. 8A shows the results of labeling cells with Nestin, Tuj-1, and GFAP (green) before the shaking step, and FIG. 8B shows the results of labeling the cells with Nestin, Tuj-1, and GFAP ( Green). In Figure 8, the bar is 100㎛. 8A and 8B, Merge is a diagram showing the result of staining with DAPI and a diagram showing the result of labeling with Tuj-1 (first row), Nestin (second row), or GFAP (third row). It is shown by merging.
9 shows the results of observing with a fluorescence activated cell sorter (FACS) after separating retinal ganglion cells according to an embodiment of the present invention. Specifically, FIGS. 9A and 9B show flow cytometry histograms for signals of Nestin, Tuj-1, and GFAP, respectively, appearing in cells before and after the shaking step.
10A and 10B show the results of multivariate flow cytometry for signals of Nestin/Tuj-1 and Nestin/GFAP, respectively, appearing in cells before and after the shaking step.
A1 and A2 of FIG. 11 are the number and ratio of Tjul-1 positive cells measured at different times in the shaking treatment with an orbital shaker, and B1 and B2 of FIG. 11 are measured by varying rpm in the shaking treatment with an orbital shaker. It is the number and percentage of Tjul-1 positive cells.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Retinal ganglion cell marker and suitable retinal tissue selection

In all animal experiments, the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) and Institutional Animal Care and Use Committee (IACUC) for Use of Animals in Ophthalmic and Vision Research were followed.

1-1. Selection of markers to identify retinal ganglion cells

Thy-1, Tuj-1, Nestin and GFAP were used to select suitable markers for RGC identification.

Thy-1 is a representative T cell marker, and is also detected in epithelial cells, dendritic cells in the epidermis, neurons, and the like. Nestin is a type of type VI intermediate filament protein and is used as a marker for neuroepithelial stem cells. Tuj-1 is used as a vascular marker of the nervous system expressed in differentiated cells such as neurons. GFAP is widely used as a marker for retinal astrocytes and Muller cells.

To determine the optimal RGC marker, immunohistochemistry experiments were used to determine the 2nd day (P2), 7th day (P7) and 14th day (P14) SD (Sprague Dawley®) rat (Orient Bio, Korea). ) Were examined for expression of Thy-1, Tuj-1, Nestin and GFAP in the retinal tissues.

Rat retinal sections of P2, P7 and P14 were prepared. First, the eyes were separated, fixed in 4% paraformaldehyde, and dehydrated by putting them in 60, 70, 80, 90, or 100% alcohol. The dehydrated tissue was put in xylene and then embedded in paraffin. It was cut (Section) using a microtome (Leica RM2235; Leica Microsystems, Bensheim, Germany) to a thickness of 5 μm and placed on Superfrost ^TM plus microscope slides (Thermo Fisher Scientific, Pittsburgh, PA, USA).

P14 thymus cell (T cell) section was prepared in the same manner as the retinal section.

Rat retinal sections of P2, P7, and P14 and P14 thymic cell sections were fixed with 4% paraformaldehyde. At room temperature, tissues and cells were incubated for 1 hour in a blocking solution containing 1% normal goat serum (Jackson ImmunoResearch, PA, USA) and 0.1% Triton X-100. Thereafter, tissues and cells at room temperature were treated with anti-Thy-1, Alexa Fluor® 488-conjugated anti-nestine (Abcam, Cambridge, UK), anti-GFAP (glial fibrillary acidic protein, Abcam, Cambridge, UK) and anti- Tuj-1 (Sigma-Aldrich, MO, USA) antibody was treated for 2 hours. Anti-GFAP and anti-Tuj-1 antibodies were visualized using Alexa Fluor® 488-conjugated anti-rabbit IgG antibody (Abcam) .

To visualize the nuclei, all tissue samples and cells were counterstained with 10 μg/ml of DAPI for 10 minutes at room temperature. Images were captured using a Nikon ECLIPSE Ti-s fluorescence microscope system (Nikon, Tokyo, Japan).

1 and 2 show the results of labeling the retinal tissues and thymus cells of P2, P7, and P14 rats with Thy-1 (green) through immunohistochemistry experiments. As a positive control, rat thymocytes were labeled with anti-Thy-1 antibody, and nuclei were counterstained with DAPI. GCL is the ganglion cell layer, IPL is the inner plexiform layer, NBL is the neuroblast layer, INL is the inner nuclear layer, and OPL is the outer plexiform layer. ), ONL represents the outer nuclear layer, IS/OS represents the photoreceptor inner segment/outer segment, and RPE represents the Retinal Pigment Epithelial Cell. Do).

As shown in FIGS. 1 and 2, Thy-1 was mainly expressed in P14 rat retinal tissue, the amount thereof was small in P7 rat retinal tissue, and not expressed in P2 rat retinal tissue, so Thy-1 is a retinal ganglion cell. Was not suitable as a marker for discriminating.

3 and 4 show the results of labeling the retinal tissues of P2, P7, and P14 rats with Nestin, Tuj-1, and GFAP. In FIG. 3, nestin, Tuj-1 and GFAP appear as green signals, and in FIG. 4, nestin appears as green and Tuj-1 as red signals, and the nuclei were counterstained with DAPI. 3 and 4, nestin and Tuj-1 were expressed in the inner reticular layer (IPL) and ganglion cell layer (GCL) of the P2, P7, and P14 rat retinal tissue. On the other hand, GFAP was weakly observed in P7 and P14 rat retinal tissues, but not in P2 rat retinal tissues.

Based on the above results, the expression of Nestin and Tuj-1 in rat retinal tissue was used as an indicator of RGC identification.

1-2. Selection of retinal tissue suitable for separating and concentrating retinal ganglion cells

The immunohistochemistry experiment for CD-4 was the same as in Example 1-1, except that Alexa Fluor® 488-conjugated anti-rabbit IgG antibody (Abcam) was used.

5 shows the expression of CD4 (green) in the retinal tissues and lymph nodes of P2, P7 and P14 rats. As shown in FIG. 5, CD4 was partially expressed in the rat retinal tissues of P7 and P14, but was not expressed in the rat retinal tissues of P2, and CD4 was expressed in the lymph nodes used as a positive control. From this, it was found that there were no immune cells (eg, macrophages or microglial cells) in the retina of the P2 rat. Therefore, the retinal ganglion cells were isolated using the retina of a P2 rat.

Example 2: Isolation and culture of retinal ganglion cells (RGC)

In order to isolate the retinal ganglion cells (hereinafter, RGC), 15 SD rats (P2) of the second day of life (Orient Bio, Korea) were used. Since the retinal ganglion cells are small in size as the retinal ganglion cells are not differentiated from the retinal tissue, the second-day rat can easily be separated by a cell filter and survive a relatively long period after culture. 6 shows a process of separating retinal ganglion cells according to an embodiment of the present invention.

To prepare the separation of the retinal tissue, a flask for tissue culture was coated with laminin (Santacruz Biotechnology, INC., TX, USA) diluted in PBS (phosphate-buffered saline) at 3.5 µg/cm ² and incubated at room temperature for 1 hour. . After that, the coated flask was washed with PBS and stored at 4°C.

Retinal tissue was separated from the eyeball of P2 rat, and the separated tissue was separated in 20 ml of HBSS (Hank's balanced salt solution; Life Technologies, Grand Island, NY) containing 5 ml of 0.25% trypsin-EDTA at 37°C for 10 minutes. During incubation. Thereafter, the tissue was softened by pipetting, and the solution containing cells was mixed with 25 ml of complete media, and the obtained solution was mixed with a 40 μm cell strainer; SPL Life Sciences, Gyeonggi-do, Korea ) Through. Cells were collected and centrifuged at 1,500 rpm for 3 minutes, and then 10% fetal bovine serum (FBS; Life Technologies), 1% insulin-transferrin-selenium (ITS; Thermo Fisher, MA, USA), 1 ng/ml brain-derived neurotrophic Resuspended in NB medium (neurobasal medium; Life Technologies, USA) containing factor (BDNF, Peeprotech, NJ, USA) and 1% penicillin-streptomycin. Approximately 1× ^{10 8} cells were collected.

The collected cells were inoculated into a laminin-pre-coated 25 cm ² flask (T-25 flask; SPL Life Sciences) at a concentration of about 2.5 x 10 ⁷ cells per flask, and 37°C and 5% CO _It was cultured under complete medium in a wet incubator of ₂ . After 5 days, the flask in which the mixed retinal cells were attached and cultured was shaken for 2 hours at 200 to 300 rpm using an orbital shaker (FINEPCR, Gyeonggi-do, Korea).

By the shaking step through an orbital shaker, some cells were attached to the flask and some cells were released from the flask according to the difference in binding affinity for laminin. The culture medium containing the separated cells from the flask was separated, and centrifuged at 1500 rpm for 3 minutes to obtain about 3 x 10 ⁵ retinal ganglion cells.

Thereafter, the isolated cells were re-inoculated in a flask coated with new laminin at a concentration of about 1×10 ⁵ retinal ganglion cells. Retinal ganglion cells (RGCs) were additionally cultured for 7 days in an incubator moistened with 37°C and 5% CO ₂ to maintain morphology and function, and complete media was replaced every day. I did.

Example 3: Confirmation of Isolation and Enrichment of Retinal Ganglion Cells by Shape

The morphology of the cells before performing the shaking step and the cells concentrated after culturing for 7 days after the shaking step was confirmed through a phase contrast microscopy (Nikon Eclipse Ti). 7A shows the cells before the shaking step and FIG. 7B shows the cells after culture for 7 days after shaking.

As shown in FIG. 7A and 7B, the cells separated by shaking by the method of Example 2 and cultured for 7 days were round nucleus, stretched-out axon, and a number of It was confirmed that the characteristics of retinal ganglion cells, such as dendrite, are well displayed. Through this, it was found that the separation and concentration of retinal ganglion cells proceeded smoothly.

Example 4: Confirmation of Isolation and Enrichment of Retinal Ganglion Cells by Markers

Using the markers of retinal ganglion cells (Tuj-1 and Nestin) selected in Example 1, it was confirmed whether the retinal ganglion cells (RGC) were isolated and concentrated by the method of Example 2. In flow cytometry and immunocytometry, the degree of isolation and enrichment of retinal ganglion cells was determined through Tuj-1-positive, nestin-positive and GFAP-negative signals.

4-1. Isolation confirmation of retinal ganglion cells by immunohistochemistry experiment

Each of the cells before and after the shaking step of Example 2 were labeled with anti-Tuj-1, anti-nesin and anti-GFAP antibodies, and the same as the method of Example 1-1. Chemical experiments were carried out.

8A and B show the results of immunohistochemical staining of cells before and after the shaking step, respectively. 8A is a result of labeling cells with nestin, Tuj-1, and GFAP (green) before the shaking step. 8B is a result of labeling cells with Nestin, Tuj-1, and GFAP (green) after the shaking step.

8A and 8B, in the cytoplasm of an extended neurite (especially, a neurite having a long axon) and a round cell body of a retinal ganglion cell (RGC), Tuj- 1 and Nestin were expressed. After the shaking step, the number of cells that were not stained with the signals of Tuj-1 and Nestin decreased significantly. The expression of GFAP, a marker of astrocytes and Mueller cells, was observed before the shaking step, but the expression of GFAP was not observed after RGC was purified through the shaking step using an orbital shaker.

The measured amounts of Tuj-1 and Nestin, markers of retinal ganglion cells, did not differ significantly after shaking, and GFAP, a marker of astrocytes and Mueller cells, was not measured after shaking. The cells were isolated, and it was found that the retinal ganglion cells were isolated and enriched.

4-2. Confirmation of enrichment by flow cytometry analysis

Flow cytometry was performed to quantify the purity or enrichment of retinal ganglion cells according to the method of Example 2 above.

Cells isolated by the method of Example 2 were immunostained with anti-Tuj-1, anti-nestine, and anti-GFAP antibodies, and analyzed with a fluorescence-activated sorter (FACS). Specifically, the cells were washed with a flow-cytometry staining buffer (PBS containing 1% FBS, 3% BSA, and 0.02% sodium azide). Cells were treated with Alexa Fluor® 488 conjugated anti-Nestin antibody (Abcam), FITC conjugated mouse IgG1 antibody, kappa-isotype control (Abcam), anti-Tuj-1 (Sigma-Aldrich) and anti-GFAP in flow cytometry buffer. Incubated with antibody (Abcam) at 4° C. for 30 minutes. Anti-Tuj-1 and anti-GFAP antibodies were visualized by Alexa Fluor® 647-conjugated AffiniPure F(ab')2 fragment anti-rabbit IgG (Jackson ImmunoResearch). Labeled retinal cells were obtained and analyzed based on area and aspect ratio using BD FACS CaliburTM (BD Biosciences, CA, USA). Data were analyzed using FCS Express (De Novo Software, CA, USA).

9A and 9B show flow cytometry histograms of signals of Nestin, Tuj-1, and GFAP before and after the shaking step of Example 2, respectively. Referring to FIG. 9A, the proportions of Nestin, Tuj-1, and GFAP positive cells were 42.92%, 41.98%, and 60.27%, respectively, before the shaking step. Referring to FIG. 9B, after the shaking step, the proportion of nestin and Tuj-1 positive cells increased to 94.56% and 91.02%, respectively, while the proportion of GFAP positive cells decreased to 21.72%. After the shaking step, the proportion of nestin and Tuj-1 positive cells increased by 51.64% and 47.04%, respectively, and that of GFAP positive cells decreased by 38.55%.

In addition, multiple parameter flow cytometry was performed on both Nestin and Tuj-1 labeled RGCs. To select cells, cells and debris that were killed by forward and side scatter were excluded, and alignment windows were set for positive and negative signals. The results before and after the shaking step of Example 2 were compared.

10A and 10B show the results of multivariate flow cytometry analysis of differences in signals between nestin/Tuj-1 and nestin/GFAP appearing in cells before shaking and cells after shaking, respectively. As shown in A and B of Fig. 10, the proportion of cells expressing Tuj-1 and nestin simultaneously (assumed as RGC) (blue area) before the shaking step was 46.79%, and 86.31% after the shaking step. In addition, when the isolated cells were double-labeled by nestin and GFAP, the proportions of nestin-positive and GFAP-negative cells (purple regions) assumed to be RGC were 63.88% and 85.47%, respectively, before and after the shaking step, respectively. It was found that the purity of retinal ganglion cells (RGC) increased by 21.59% by the shaking step.

Example 5: Confirmation of RPM and treatment time of shaking

In order to confirm the optimal conditions for separating the retinal ganglion cells in the shaking step, the experiment was performed at different RPMs and treatment times. Other experimental conditions were the same as in Example 2.

A1 of FIG. 11 shows the amount of Tjul-1 positive cells released according to the shaking treatment time, and A2 of FIG. 11 shows the ratio of the released Tjul-1 positive cells according to the shaking treatment time. Referring to A1 and A2 of FIG. 11, as the shaking treatment time increased, the amount of released Tjul-1 positive cells increased, and the proportion of Tjul-1 positive cells did not decrease. Since Tjul-1 positive cells are retinal ganglion cells, it was found that the most efficient separation of retinal ganglion cells was achieved when the shaking treatment time was 3 hours.

B1 of FIG. 11 shows the amount of Tjul-1 positive cells released according to the shaking treatment with different rpm, and B2 of FIG. 11 shows the ratio of the separated Tjul-1 positive cells according to the shaking treatment with different rpm. will be. Referring to B1 and B2 of FIG. 11, when the rpm is 100 to 300, the proportion of Tjul-1 positive cells all showed a high ratio and there was no significant difference. Therefore, it was found that the retinal ganglion cells can be efficiently separated in the range of 100 to 300 rpm.

Claims (8)

Obtaining cells from retinal tissue;
Inoculating and culturing the obtained cells in a container coated with a cell adhesion protein;
Shaking the container after incubation; And
Comprising the step of collecting the released cells from the container,
The cell adhesion protein is laminin,
How to separate retinal ganglion cells.
The method of claim 1,
The method for separating retinal ganglion cells wherein the retinal tissue is isolated from a rat or mouse.
The method of claim 2,
The method of separating retinal ganglion cells that the rat is within 2 days of age.
delete The method of claim 1,
The shaking step is a method of separating retinal ganglion cells that are performed for 2 to 3 hours at 100 to 350 rpm using an orbital shaker.
The method of claim 1,
The step of obtaining cells from the retinal tissue,
Preparing retinal tissue as an aqueous cell solution;
Filtering the aqueous cell solution with a cell filter; And
A method for separating retinal ganglion cells comprising the step of collecting the filtered cells.
delete delete

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