Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

tified that provide further information useful for the design and evaluation of experiments using retinal neuron cultures.

II. DESIGN CONSIDERATIONS

A.Culture Type

The type of culture chosen is often constrained by the cellular response that is to be studied. For example, the effects of experimental treatments on retinal neuron survival and differentiation are easily studied in monolayer cell cultures. In these cultures, a completely dissociated retinal cell suspension is placed in tissue culture dishes, and within 6 to 8 hours, the neurons attach to the dish surface and begin to differentiate neuritic processes. Because each neuron can be directly inspected using an inverted microscope, it is relatively easy to determine whether the neuron is alive and whether process growth has been altered. These features also can be studied in explant retinal culture systems in which some of the original retinal tissue organization is retained. However, because it is often difficult to inspect individual living neurons in explants, assessment of survival and differentiation can be better accomplished using appropriate biochemical or histological analyses.

B.Developmental Age

The developmental age of the retina also can be important. For example, growing cells from embryonic retina cells are often easier to maintain in culture than cells from adult retina. It is also possible to expand the number of embryonic retinal cells by encouraging cell division. In contrast, cells from older embryo retina or adult retina typically are post-mitotic. Hence, it can be challenging to obtain large numbers of retinal cells for culture experiments from adult retina. However, such experiments can be important because the cellular responses of embryonic retinal cells can vary from the responses of adult retinal cells.

C.Culture Media

The various culture media contain different types and amount of salts, amino acids, energy sources, buffers, and vitamins. For example, Dulbecco’s modified Eagles medium (DMEM), which can be used for culturing embryonic and newborn rat or mouse retinal neurons, contains 7 salts, 13 amino acids, 8 vitamins, glucose, and pyruvate. In contrast, Medium 199, which has been used for culturing chick retinal neurons, contains 11 salts, 21 amino acids, 17 vitamins, and glucose, plus 17 additional ingredients, but no pyruvate. For the successful culture of retinal neurons expressing glutamate or aspartate receptors, it may be beneficial

to eliminate these amino acids from the medium because they could induce excitotoxic stress. Neurobasal medium (available from Gibco BRL) is one such medium that has been formulated without glutamate or aspartate [14]. It is important to note that the pH of tissue culture medium often is regulated by exposing the medium to an atmosphere containing a specific amount of carbon dioxide. For example, DMEM is designed to buffer correctly in an atmosphere containing 10% carbon dioxide. However, media such as Ham’s F12 and RPMI 1640 require 5% carbon dioxide. The correct concentration of carbon dioxide for a particular medium can be obtained from the medium supplier or from previously published protocols. As each medium has its advantages and limitations, the best medium for each retinal neuron type should be determined empirically.

D. Media Supplements

The performance of media is often enhanced with supplements including hormones, growth factors, cofactors, lipids, carrier proteins, and natural fluids. Hormones that often are beneficial include insulin, progesterone, and hydrocortisone. Growth factors that have been helpful for the maintenance of certain retinal neurons include fibroblast growth factor–2, nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, neurotrophin-3, and neurotrophin-4 [3,15,16]. Useful cofactors, molecules that facilitate certain biological reactions, include compounds such as sodium selenite and copper sulfate. Lipids that have been used in certain retinal neuron cultures include linoleic acid and docosahexaenoic acid [10,17]. Carrier proteins that have proved to be beneficial include transferrin and serum albumin. Note that the addition of unbuffered solutions of certain supplements can change the media pH. Natural fluids such as sera, amniotic fluid, and cerebrospinal fluid have been used as supplements. These contain hormones, growth factors, and other potentially beneficial molecules. It should be noted that the composition of natural fluids can vary among suppliers and even among lots from the same supplier. With natural fluids, as with purified supplements, the optimal amount to add can vary among retinal neuron types and culture systems. Hence, the best mixture of supplements and/or natural fluids needs to be determined for each case.

E.Substrate Enhancements

The performance of retinal cultures often can be improved by application of specialized coatings to the culture dishes. The first type of coating is the charge modifiers and includes poly-L-lysine and poly-L-ornithine. Many retinal neurons express glycoproteins that contain negatively charged sialic acid. As a result, they do not bind strongly to negatively charged tissue culture plastic. Poly-L- lysine and poly-L-ornithine are positively charged synthetic amino acid polymers.

Coating the culture dish with either of these compounds changes the charge of the tissue culture surface and thereby promotes neuronal attachment [18]. The second type of coating includes extracellular matrix molecules such as collagens, fibronectin, laminin, and heparin sulfate proteoglycan. These molecules can bind to specific cell surface receptors, facilitate growth factor action, and can often dramatically improve neuronal differentiation and survival [19,20]. The third type of coating includes molecules with specific affinities such as antibodies and lectins. These molecules can facilitate or modulate binding of certain subsets of cells present within a dissociated retinal cell suspension [18]. As with media and supplements, the optimal coating should be determined for each retinal cell type and culture system.

F.Co-Cultures

The survival of certain retinal cell types has been improved by co-culturing with other cell types such as astrocytes, or with target neurons such as lateral geniculate neurons in the case of retinal ganglion cells [6]. In the case of astrocytes, a confluent monolayer of astrocytes is often generated prior to seeding the retinal neurons (adding the dissociated cell suspension to the cultures). Because retinal neurons can grow on top of the astrocytes in these cultures, their survival and differentiation may be easily evaluated with the inverted microscope. Retinal neuron aggregate cultures also can be generated in the presence of other cell types. This will be further discussed below. Co-cultures have been useful for investigating the cellular basis of beneficial cell interactions.

G.Cell Density

The density of cells placed within monolayer cell cultures can influence cell survival and differentiation [21]. Usually, increased survival is observed with increased cell density. This is because the retinal neurons produce molecules that are beneficial for their survival in vitro. If cell density is too high, however, differentiation can be inhibited. Also, cell clumping can become problematic in high-density cultures. This can make it difficult to assess survival changes and changes in cell structure.

III. SELECTED METHODS

A.Isolation of Chick Retina

This protocol is appropriate for chick embryos ranging in age from 7 to 12 days. All instruments must be sterilized before use. After being briefly wiped with 70% ethanol, the rounded end of the egg is opened using blunt forceps, and the shell

membrane over the embryo is removed with fine forceps. Using fine-curved forceps, the vascular connections to yolk membranes are broken. Next, the embryo is lifted out using blunt-curved forceps and placed in a 60 mm dish. After separation of the head, the eyelid rudiments are either pulled off with fine forceps or removed using spring-action scissors. Then the eyes are lifted from their sockets using the curved portion of curved sharp forceps and transferred to a 60-mm dish containing Hank’s buffered saline solution (HBSS). Any orbital tissues such as muscle and fat tissue is then removed from the surface of the eyes.

Next, the tips of sharp forceps are inserted into the globe near the optic nerve to create a small opening. With two pairs of sharp forceps, the edge of the sclera at the opening is grasped at opposite sides of the opening and, by spreading, the sclera and retinal pigment epithelium (RPE) are peeled from the globe—like removing a thin glove from a hand. The lens and cornea typically come off with the sclera/RPE. The center of the vitreous gel is grasped with blunt forceps, and the retina is gently teased from the vitreous gel using sharp forceps. The retina is transferred to another 60 mm dish containing HBSS by gently lifting with either fire-polished glass hooks or spread curved forceps. Nonretinal tissue present on small portions of the retina are sliced off using sharp sterile tungsten needles that have been glued with epoxy to glass rod handles. The cleaned retina is transferred to another 60 mm dish containing HBSS. At this point, the retina is ready to be sliced for sliced cultures, diced with tungsten needles and then placed in explant cultures, or diced and then enzymatically dissociated into a single cell suspension.

This protocol can be adapted to newborn and adult rat eyes by using appropriate sterile surgical technique to remove eyes and then proceeding as described to isolate the retina.

B. Generating Retinal Neuron Monolayer Cultures

This method is adapted from a protocol for retinal neuron cultures that promotes the survival and differentiation of several types of retinal neurons [10]. Precoat tissue culture dishes with a charge modifier (e.g., by filling 35 mm dishes with 0.1 mg/mL 30–70 kDa poly-L-ornithine dissolved in purified water and sterile filtered; incubate at least 1 h at room temperature). Add an extracellular matrix coating (e.g., 0.5 g/mL laminin dissolved in DMEM; incubate in 10% CO2 incubator at 37oC, overnight). Prepare media fresh, add supplements (e.g., Medium 199 containing 10% heat-inactivated fetal bovine serum [FBS], 110 g/ mL linoleic acid–albumin, 100 U/mL penicillin, and 2 mM glutamine) and place in humidified incubator (5% CO2, 37°C) to equilibrate temperature and pH.

Isolate chick retina and dice into small pieces (1 mm squares) as described above. The retinal pieces are transferred to a test tube and allowed to settle to the bottom. The HBSS is replaced with calciumand magnesium-free HBSS (CMF-HBSS) and incubated for 10 minutes in a 37°C water bath. After two

rinses (changes) in CMF-HBSS, a solution containing 0.25% trypsin and 1.85 U/ml DNAse (dissolved in CMF-HBSS) is added and the tissue is incubated at 37°C for 20 min. After trypsin is removed, the retinal pieces are rinsed with HBSS containing 10% fetal bovine serum (FBS). After 1 mL of 10% FBS/HBSS is added, the tissue is triturated (sucked in and out of a Pasteur pipette in which the tip has been reduced by flame polishing) several times until it dissociates into a single cell suspension. Avoid excessive trituration that can reduce viability. Examine a drop of the suspension placed on a microscope slide using a microscope with phase optics. If there are cell clumps present, these can be removed by passing the cell suspension through a sterile Nytex filter fabric affixed to a sterile glass tube.

The concentration of cells in the suspension is determined by adding one drop of suspension to each side of a Neubauer ruling hemacytometer. After placing the hemacytometer on a conventional (upright) phase microscope, cells visible in a 1 mm square area are counted (a group of 16 squares present at the corners of the ruling). A button-activated counter facilitates tallying the count. Multiply the count obtained by 10,000 to obtain cells per milliliter. Total yield is calculated by multiplying the suspension concentration by the total suspension volume. After appropriate dilution with final media, the cell suspension is added to the culture dishes. In the case of chick retinal neurons seeded in 35 mm dishes described above, dilute the suspension to 400,000 cells/mL and add 1 mL per dish (add to the 1 mL already present in the dish). The dishes are then placed into the incubator.

IV. ALTERNATIVE CULTURE FORMATS

A.Retinal Explant Cultures

Retinal explant cultures can be useful for examining neurite outgrowth as well as neuronal differentiation [19,22,23]. Typically, isolated retina is diced into pieces and the pieces are placed in a drop of medium containing 40 to 50% serum on the bottom of a culture dish. After allowing the serum to clot, which holds the explant in place, add culture medium. Within 1 to 2 days, glia are observed growing out of the explant. Several days later, neurites are observed radiating from the explants. These cultures have been used to evaluate interactions between growing axons and extracellular matrix, the effects of growth factors, and the role of glia in axon extension.

B.Retinal Slice Cultures

Cellular contacts and interactions are typically well preserved in slice cultures [24,25]. The usual approach is to lay the isolated retina on a cellulose filter mem-

brane and transfer it to the sterilized stage of a tissue chopper, where slices 100– 400 µm thick are cut. The slices, along with the attached membrane, are then transferred into culture medium in a culture dish and placed into an incubator. These cultures preserve the association of retinal cells with their microenvironment and neighboring cell types. Within the slice, synaptic associations also are retained. Cells within retinal slices can be studied using electrophysiological recordings. Subsets of cells, such as amacrine cells, can be identified and studied within these slice cultures using specific immunohistochemical markers.

C. Reaggregation Cultures

The presence of adhesionor contact-mediated interactions can be studied in reaggregation cultures [26–28]. A retinal cell suspension is prepared as described above. Aliquots of the suspension are transferred to reaggregation culture dishes or small (e.g., 25 mL), sterile Erlenmeyer flasks and then placed in a gyratory shaker contained within a 37°C incubator. The size of the aggregate is dependent on cell density, gyration amplitude (10–25 mm), and gyration frequency (50– 80 rpm). By mixing the retinal cell suspension with other cell type suspensions (such as from lateral geniculate), target cellular interactions can be studied. Reaggregate cultures can be evaluated by biochemical analysis, by counting, or by histology.

D. Enriched Photoreceptor Cultures

Methods have been developed to generate mixed retinal neuron cultures from embryonic chick or mouse eyes that contain identifiable photoreceptors as well as multipolar neurons (neurons that express multiple neurites) [10,29]. The photoreceptors are readily identified by their extension of a single neurite, their expression of opsin, and, in the case of chick photoreceptors, a cytoplasmic lipid droplet visible by phase microscopy. Adding 2 mM kainic acid or 1–2 nM β-bungaro- toxin to the cultures can eliminate up to 70% of the multipolar neurons without apparent effect on the photoreceptors [30,31]. Combined treatments with these agents does not show additive effects or potentiation between the toxins [31]. Another method useful for generating cultures of purified photoreceptors separates these cells from other retinal neurons during isolation through the use of vibratome sectioning [32].

E.Purified Retinal Ganglion Cell Cultures

Retinal ganglion cells can be purified from retinal cell suspensions using cell panning [33] and then cultured in chemically defined media [2]. This technique relies on retinal ganglion cell expression of the cell surface marker protein Thy-

1 and the absence of expression of an antigen recognized by anti-macrophage antibody. A retinal cell suspension is generated as described above. This suspension is incubated with monoclonal mouse–anti-macrophage antibodies and then poured into a large petri dish precoated with goat–anti-mouse IgG antibodies. Nonadherent cells are collected and then incubated in a second petri dish precoated with anti–Thy-1 antibody. After repeated washing with HBSS to remove nonadherent cells, the remaining cells are greater than 95% retinal ganglion cells. The attached cells are then released from the panning dish using mild trypsin digestion and placed in monolayer cell culture.

V.TROUBLESHOOTING

A.Pathogen Contamination

The appearance of yeast, fungi, and bacteria in cultures will alter results and eventually destroy the cultures. Yeast infection appears as a proliferation of round or ovoid particles within the cultures. Fungi form filamentous threads (mycelia) that often are branched. Bacteria appear as clumps of small rods or particles. Excellent pictures of cultures infected with these microbes are present in a cell culture manual by Freshney [34]. Mycoplasma also can infect cultures and reduce viability. It is not possible to see mycoplasma by microscopic inspection. However, they can be detected by staining cultures using the dye Hoechst 33258 or by use of a commercial test kit (available from Gibco BRL). Antibiotics rarely can control an established infection. Usually, the best course of action is to discard the contaminated cultures and suspected media, resterilize the equipment and workspaces of the culture laboratory, and then resume with new media and plates.

B.Chemical Contamination

Abnormal performance of solutions used in tissue culture procedures can reflect chemical contamination. For example, if glassware used to prepare media is insufficiently rinsed after washing, residual detergent can be left on the glassware walls and then contaminate solutions placed in the glassware. A failure in the performance of the laboratory water purifier also can introduce chemical contamination. Other possible sources of chemical contamination include a pH electrode that is inadvertently used for measuring nontissue culture solutions, and spilled chemicals on the pan of the laboratory microbalance used to weigh purified media supplements.

C.Inactivation of Tissue Culture Reagents

Several tissue culture reagents have a relatively short shelf life. For example, glutamine in solution becomes deactivated within several days. Hence, frozen

stock glutamine often is added to media just before use. Certain peptide supplements can become deactivated by repeated freeze-thaw. Hence, they should be stored in a non–frost-free freezer. Certain enzymes, such as trypsin, can become deactivated by auto-digestion if kept at room temperature or 37°C for an extended period. Hence, frozen enzyme solutions used in tissue dissociation should be thawed in the water bath just before they are needed.

D. Incorrect Incubator Settings or Function

Incorrect concentration of carbon dioxide within the incubator will produce incorrect pH within the media. As mentioned above, the correct carbon dioxide concentration for a particular media can be obtained from the supplier. Correct performance of the incubator controls can be confirmed using a Fyrite flue-gas analyzer (available from Fisher Scientific). A small air sample is withdrawn from the incubator chamber through a special port without opening the incubator door. This air sample is exposed to an alkali solution that absorbs the carbon dioxide. The change in the volume of the air sample is measured to determine carbon dioxide concentration in the original sample. Regular testing of incubators will often identify performance problems prior to the loss of valuable experiments.

E.Phototoxicity

Examination of cultures using an inverted microscope is often the best way to monitor the status of retinal monolayer, explant, or slice cultures. However, the cells are easily damaged by too much light exposure. Use of a green filter can reduce the more-harmful short wavelength light and thus protect the cultures during examination.

VI. RECOMMENDED RESOURCES

Excellent books on basic cell culture technique are available that discuss the culture laboratory, aseptic technique, selection of culture supplies, sterilization, basic considerations in the generation of primary cultures, characterization of cell cultures, and contamination control [34,35]. A classic reference for the formulation of chemically defined media for neuronal cultures is a book series edited by Barnes, Sirbasku, and Sato [36]. Also helpful are several articles in recent editions of Methods in Cell Biology [37–45].

VII. CONCLUDING COMMENTS

First, start with an established protocol, if available. If one that accomplishes what you want is not available, start with the most similar one you can find and