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Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Neuroscience
 

Summary

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

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O'Meara, R. W., Ryan, S. D., Colognato, H., Kothary, R. Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues. J. Vis. Exp. (54), e3324, doi:10.3791/3324 (2011).

Abstract

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines.

Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy & de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies.

While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

Protocol

Ethics Statement

The mice used in this work were cared for according to Canadian Council on Animal Care (CCAC) guidelines. Ethical approval for experiments conducted was obtained from the University of Ottawa Animal Care Committee under protocol number OGH-119.

1. Dissection – neonatal mouse cortex for OPC extraction

  1. Sacrifice P0-P2 mouse according to institutional guidelines.
  2. Dissect the brain and place in a Petri dish containing ice-cold MEM (antibiotic-free).
  3. Transfer the dish to a dissection microscope.
  4. Using a scalpel with the brain dorsal side up, make a shallow incision sagittally along the most medial edge of each cortex (Fig 1a). This incision should only pass through the meningeal layer in order to facilitate its removal.
  5. Use fine tipped forceps to peel off the meninges in a lateral fashion. If done carefully, this layer can be removed in one piece. During this step, remove the olfactory bulbs.
  6. With the brain ventral side-up, make a deep sagittal incision where the cortex meets the ventral area of the diencephalon (Fig 1b).
  7. With the brain dorsal side-up, separate the cortices from the midbrain by prying the tissue in a medial to lateral fashion (Fig 1c, c'). Remove any residual meninges at this step.
  8. Dice each cortex into approximately 4 pieces and gently transfer to a 15 mL conical tube containing 350 μL of MEM per mouse brain. Keep the tube on ice until all mice have been processed.
  9. Repeat steps 1.1-1.8 for remaining mice.

2. Dissociation of neonatal cortices and maintenance of mixed glial cultures

Note: The introduction of bubbles into the cell suspension should be avoided during all of the following steps.

  1. Add the 15 mL conical tube containing the freshly dissected brains to a 37°C water bath for 3 min.
  2. Transfer brains to a sterile tissue culture hood.
  3. Gently pass diced cortices through a P1000 pipette tip to generate smaller fragments. Stop pipetting once there are no brain pieces large enough to disrupt a smooth flow of suspension through the pipette tip.
  4. Add 75 μL of OPC papain solution per brain into the conical tube. The OPC papain solution must be pre-warmed at 37°C for 20 min prior to use.
  5. Incubate in a 37°C water bath for 20 min. Approximately every 2 minutes, gently invert the tube to prevent tissue aggregation. During this time, add 5 mL of mixed glial culture media to each poly-L-lysine (PLL) coated (1 mg/mL) T25 flask (one flask per mouse brain), and place in a 37°C tissue culture incubator at 8.5% CO2.
  6. After 20 min, return the tissue suspension to the sterile hood and add 2 mL of mixed glial culture media per brain to the tube. Let sit for 10 min at room temperature to allow inactivation of the OPC papain solution.
  7. Aliquot the tissue suspension into 5 mL plastic tubes. The number of tubes should match the number of brains dissected, resulting in approximately 2.5 mL per tube.
  8. Using a sterile flame-polished glass Pasteur pipette, gently triturate the tissue in each tube. Triturate slowly at first, and gradually increase speed as pieces dissociate. Triturate approximately 10-15 times, however this number may vary based on the efficacy of the digestion.

Note: Under-trituration will result in poor dissociation of the tissue, whereas over-trituration will negatively impact on cell viability. It is important to not introduce bubbles into the solution, as this will severely impact cell viability.

  1. Once there are no visible tissue clumps remaining in the suspension, transfer to a 50 mL conical tube containing 4 mL of mixed glial culture media per brain (i.e., 4 brains = 16 mL mixed glial culture media).
  2. Gently invert the 50 mL conical tube and repeat for the remaining 5 mL tubes.
  3. Aliquot the pooled cell suspension into 15 mL conical tubes (approximately 6.5 mL per 15 mL tube). The number of 15 mL tubes should match the number of brains dissected.
  4. Centrifuge the tubes at 1200 rpm (~300 g) for 5 min.
  5. Carefully aspirate the supernatant and add 1 mL of warm mixed glial culture media to each 15 mL conical tube.
  6. Slowly resuspend the pellet with a P1000 pipette tip, being careful not to introduce bubbles. Add the cell suspension from each tube to a pre-equilibrated PLL-coated T25 flask, rendering the total volume of the culture media to 6 mL.
  7. Place the flasks in a tissue culture incubator for 3-4 hours to allow the cells to attach to the PLL substrate. Perform a full media change by pipetting out the media, and adding 6 mL of fresh mixed glial culture media to the flasks. This step removes much of the debris caused by the trituration, and promotes culture viability. If OL/DRGN co-cultures are desired, refer to Section 3 of this protocol.
  8. After 3 days of culture, perform a 2/3 media change by removing 4 mL of media, and replacing with 4 mL of fresh mixed glial culture media. At this point, an astrocyte monolayer should be forming on the base of the flasks.
  9. On Day 6, perform another 2/3 media change and supplement the flasks with a final concentration of 5 μg/mL insulin. At this point, an astrocyte monolayer should be clearly visible, on top of which OPCs will be proliferating.

3. DRGN isolation

Note: To produce OL/DRGNs co-cultures, DRGNs should be established the day after mixed glial culture generation. Both culture types are grown independently, and combined after 9-10 days.

  1. Sacrifice P5-P10 mouse according to institutional guidelines.
  2. Extract the spine, and transfer to a clean Petri dish.
  3. Trim away as much muscle and bone from the spine as possible (Fig 1d, d'), as this will ease the dissection of the dorsal root ganglia (DRGs).
  4. Transfer the trimmed spine to a new Petri dish ventral side-up. Using dissection scissors and starting caudally, cut medially through the spinal column in a longitudinal fashion.
  5. Using two pairs of forceps, gently pry open the spinal column to expose the spinal cord.
  6. DRGs can be found beneath and lateral to the spinal cord. Using fine tipped forceps, gently remove the DRGs while avoiding damage to the ganglia (Fig 1e).
  7. Transfer the removed DRGs to ice cold Hank's buffered salt solution (HBSS, antibiotic-free) in a new Petri dish. The dissector should aim to extract 40 DRGs per mouse (Fig 1f).
  8. Once the DRGs have been extracted, trim the DRGs of any excessively long roots (Fig 1g, g') to minimize introduction of contaminating cells into the culture (glial cells, fibroblasts).
  9. Transfer the DRGs to a 1.5 mL centrifuge tube containing 500 μL of ice cold HBSS.
  10. Centrifuge at 1200 rpm (~300 g) for 5 min at 4°C to pellet the DRGs.
  11. Transfer the centrifuge tubes to a sterile tissue culture hood and remove the HBSS from the tubes.
  12. Add 500 μL of pre-warmed (20 min at 37°C) DRG papain solution, and incubate the tubes in a 37°C water bath for 10 min. Invert the tubes every 2 min to prevent tissue aggregation.
  13. Repeat step 3.10.
  14. Remove the DRG papain solution and add 500 μL of pre-warmed (20 min at 37°C) Collagenase A solution. Incubate in a 37°C water bath for 10 min, inverting every 2 min.
  15. Repeat step 3.10.
  16. Remove supernatant and add 1 mL of DRGN media. Invert tube several times.
  17. Repeat step 3.10.
  18. Repeat step 3.16.
  19. Coat a sterile flame-polished glass Pasteur pipette with bovine serum albumin (BSA) by pipetting a solution of 0.25% BSA in HBSS several times. The coating with BSA solution will prevent the DRGs from adhering to the walls of the glass pipette.
  20. Triturate the DRGs with the BSA-coated pipette gently at first, and with increasing intensity once clumps begin dissociating. Triturate approximately 10-15 times, however this number is dependant on the degree of digestion, and the number of DRGs per tube.
  21. Once dissociation is achieved, pass the suspension through a 50 μm filter into a sterile Petri dish containing 7 mL of DRGN media. Filtration will eliminate much of the debris from the cell suspension, although this step is not critical.
  22. Incubate the Petri dish at 8.5% CO2 for approximately 1.25 hours.
  23. Coat several 12 mm coverslips with LN2 (10 μg/mL in PBS) in a 24-well dish during this incubation time.
  24. Once the incubation is finished, observe the Petri dish under bright field. DRGNs are identified as large bodied, phase dark cells. Swirl the Petri dish gently to lift any adhered DRGNs.

Note: Many contaminating cells will have strongly adhered to the Petri dish, thereby enriching your cell suspension for DRGNs.

  1. Transfer the cell suspension to a 15 mL conical tube. Gently rinse the dish with 4 mL of DRGN media to collect any residual DRGNs. Transfer the additional 4 mL to the conical tube.
  2. Centrifuge for 5 min at 1200 rpm (~300 g).
  3. Aspirate supernatant and resuspend the pellet in 500 μL of fresh DRGN media.
  4. Calculate the number of yielded DRGNs using a hemocytometer. Be sure to only count the DRGNs, and not other cell types. DRGNs can be identified by their large spherical cell bodies.
  5. Seed 30,000–50,000 DRGNs to each LN2-coated coverslip in 1 mL of DRGN media, and place in a 37°C tissue culture incubator at 8.5% CO2 overnight.
  6. The next morning, perform a full media change by replacing the DRGN media with OL media (minus CNTF) with a final concentration of 1% Pen/Strep and 10 μM FuDR.
  7. On Days 3 and 5, perform a 3/4 media change with the same media as in Step 3.30.
  8. On Day 7, perform a full media change with OL media (minus CNTF, Pen/Strep, FuDR).
  9. On Day 9, the DRGNs should have formed an extensive neurite bed, and are now ready to be co-cultured with OPCs.

4. Purification of OPCs from mixed glial cultures for establishment of OL-enriched cultures or OL/DRGN co-cultures

  1. On Day 9 of the mixed glial culture, transfer the flasks to an orbital shaker in a 5% CO2 tissue culture incubator. Place the flasks on top of empty T25 flasks to prevent any heat generated from the orbital shaker from adversely affecting the mixed glial cultures. Allow the cultures to equilibrate to this new incubator for 1 hour.
  2. Once the flasks have equilibrated, shake the flasks at 50 rpm for 45 min. The purpose of this shake is to remove any loosely adherent contaminating cells from the monolayer.
  3. Move cells to a tissue culture hood and remove all the media from the flasks. Replace with 4 mL of fresh mixed glial culture media supplemented with 5 μg/mL insulin.
  4. Place the flasks back onto the shaker, and allow to equilibrate for approximately 3 hours.
  5. Once the flasks are equilibrated, fasten them securely to the orbital shaker, and shake the flasks for approximately 16 hours at 220 rpm (overnight).
  6. The next morning, if OLs are to be grown in the absence of DRGNs (i.e., OL-enriched culture), coat several sterile 12 mm coverslips with LN2 (10 μg/mL in PBS) for 1 hour. Transfer the coverslips to 24-well dishes, wash with PBS followed by an OL media wash. Add 1 mL of OL media to each well and equilibrate at 8.5% CO2.
  7. Equilibrate 10 cm tissue culture dishes at 5% CO2 for 30 min. One dish will be required for every 2 flasks. These will be used for the differential adhesion-enrichment of the suspended OPCs.
  8. Once the 30 min equilibration period has passed, transfer the media from the shaken flasks to the dishes. Each dish should receive media from 2 flasks, equaling approximately 8 mL of cell suspension per 10 cm dish.
  9. Incubate the dishes at 5% CO2 for 30 min, while providing a gentle nudge at the 15 min mark. This nudge will help prevent OPCs from adhering to the 10 cm dish.
  10. Once the incubation is complete, examine the dishes under bright field. OPCs are identified as small cell clumps, typically of 3-5 cells but sometimes form large aggregates resembling neurospheres. Many non-OL lineage cells should be firmly adhered to the base of the plate. Gently swirl the plates to detach any loosely adhered OPCs, and transfer the cell suspension from each plate into a 15 mL conical tube.
  11. Centrifuge at 1200 rpm (~300 g) for 5 min.
  12. Resuspend the pellet in 1 mL of OL media with a P1000 pipette tip, followed by resuspension with a P200 pipette tip.
  13. Count the cells using a hemocytometer.
  14. For enriched-OL cultures, seed 25,000 - 50,000 OPCs to each 12 mm LN2-coated coverslip in a final volume of 1 mL OL media.
  15. For OL/DRGN co-cultures, perform a full OL media (minus CNTF) change on the DRGNs from section [3], and gently add 50,000 cells from the OPC-enriched cell suspension. Take care to not disrupt the DRGN neurite bed during the addition of OPCs.
  16. Place cultures in a 37°C incubator at 8.5% CO2, and avoid removing until fixation. Murine OPCs are sensitive to changes in pH, and removal from the incubator will alter the pH of the OL media. Also of note, addition of dH2O to the empty wells surrounding the cell cultures will prevent evaporation of the culture media, thus minimizing fluctuations in the concentrations solutes within the OL media. This will provide a more consistent environment for the OPCs.

5. Processing of cultures for immunofluorescence microscopy

  1. Fix cultures with 100% methanol at -20°C for 10 min, or 3% paraformaldehyde at room temperature for 15 min.
  2. Permeabilize coverslips with 0.1% Triton-X-100 for 10 min, wash with phosphate-buffered saline (PBS) and block for 1 hour in 10% goat serum.
  3. Incubate coverslips with primary antibodies diluted in blocking solution overnight at 4°C.
  4. Wash coverslips 3 times with PBS, and incubate with Alexa-fluor conjugated secondary antibodies (Invitrogen) diluted in blocking solution for 45 min.
  5. Counterstain with 4',6-diamidino-2-phenylindole (DAPI) and wash coverslips several times with PBS.
  6. Mount coverslips in DAKO fluorescent mounting medium.
  7. Analyze slides via immunofluorescence microscopy. In this protocol, slides were analyzed with either a Zeiss Axiovert 200M inverted fluorescence microscope or a Zeiss LSM 510 META laser scanning confocal microscope.

6. Whole cell protein extraction from OL-enriched cultures

  1. Remove 24-well cultures from incubator and cool on ice for 3 min.
  2. Carefully remove media, and add 10-20 μL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X-100, with 0.1% pepstatin, aprotinin, PMSF, leupeptin, sodium orthovanadate) to each well (A minimum of 8 wells per sample is suggested).
  3. Scrape wells using a wide-bore P1000 pipette tip, and transfer the lysate to a 1.5 mL centrifuge tube.
  4. Pass the lysate through a 30½-gauge syringe approximately 15 times, and chill on ice for 30 min.
  5. Centrifuge tubes at 14,000 rpm (~20,000 g) for 15 min at 4°C.
  6. Transfer supernatant to new centrifuge tubes, and store at -80°C.

7. SDS-PAGE analysis on enriched-OL culture protein

  1. Resolve 30 μg of protein per sample in reducing buffer by SDS-PAGE on standard 12% poly-acrylamide gels.
  2. Semi-dry transfer gels onto PVDF membranes.
  3. Block membranes for 1 hour in 5% skim milk powder in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20).
  4. Incubate membranes with primary antibodies diluted in blocking solution for 1 hour.
  5. Wash membranes 3 times with TBST for 10 min.
  6. Incubate the membranes with HRP-conjugated secondary antibodies for 45 min in blocking solution.
  7. Wash membranes several times with TBST, and incubate with Amersham ECL Plus western blotting detection reagent (GE Healthcare) for 5 min.
  8. Detect protein bands with standard scientific imaging film.

8. Representative Results:

In this protocol, OPCs are expanded on an astrocyte monolayer within a mixed glial culture. This mixed glial culture is derived from P0-P2 neonatal mouse cortex. At day 1 in vitro (DIV1), the mixed glial culture contains cells with varying morphologies as seen by phase contrast microscopy (Fig 2a). At DIV3, an astrocyte monolayer begins to form on the base of the flask, and at DIV8, OPCs can be clearly observed on the monolayer surface. At DIV9, the proliferating OPCs have reached sufficient density to be purified by overnight high-speed orbital shaking. Once the purification process has been completed, the result is an OPC-enriched cell population. At DIV1-post purification, OPCs have simple morphology, extending few processes (Fig 2b). At DIV3 post purification, cells have extended a complex meshwork of processes, reminiscent of immature OLs. At DIV6 post purification, the purified OLs have flattened and projected leaflet-like membrane structures. This morphological development is typical of the in vitro maturation of OLs.

Immunofluorescence microscopy indicates the purified cells are of OL-lineage (Fig 3a). Seeded OPCs initially express chondroitin sulfate proteoglycan (NG2), and develop into myelin-associated glycoprotein (MAG) positive immature OLs within three days post seeding (Fig 3b). At DIV6, many OLs express myelin basic protein (MBP), and possess typical mature OL morphology. Percent OL-lineage cells were quantified at different time points to determine the purity of the OL-enriched cultures (Fig 3c). At DIV1 post purification, cultures are 50±14% NG2+ve OPCs, with no MAG+ve or MBP+ve OLs. This indicates the purified OL-lineage cells are in the precursor stage at seeding time, with negligible numbers of differentiated OLs. At DIV3, many OLs have differentiated into MAG+ve cells (24±5.9%) while some retain the precursor phenotype, and remain NG2+ (13±8.0%). At DIV3, a small proportion of MAG+ve cells (3.2 1.2%) are also expressing MBP. At DIV6, 20±5.9% of OLs are MAG+ve while 12±7.3% persist as NG2+ve OPCs. In addition, 21±9.3% of cells within the culture are MBP+ve OLs at this time point. SDS-PAGE analysis shows the graded expression of 2'3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) and MBP over the 6 day culture period, further demonstrating the ability of OPCs in culture to terminally differentiate into mature OLs (Fig 3d). Collectively, these data establishes this method as a means of producing an OL-enriched culture system suitable for the study of OL maturation from OPCs.

This protocol also describes methods to establish OL/DRGN co-cultures using murine-only tissue sources. However, in order to produce the co-culture, DRGNs must first be cultured alone to produce an adequate neurite network. These post-natal murine neuron cultures are grown for 9 days in low serum media with 10 μM FuDR supplementation to prevent the proliferation of contaminating fibroblasts and glial cells. Over the course of 9 days in vitro, isolated DRGNs produce a dense neurite bed (Fig 4a). This neurite bed is immunopositive for the neuronal markers neurofilament 200 (NF) and Tuj1 (Fig 4b). At this point, purified OPCs may be added to the neurite beds, and cultured for an additional 6 days to produce myelinating co-cultures.

At DIV6 of OL/DRGN co-culture, many MBP+ve OLs can be observed among the NF+ve DRGN neurites (Fig 5a). Upon closer examination, OLs are evidenced to make contact with numerous DRGN neurites, often ensheathing them with an MBP+ve membrane (Fig 5b,c).

Figure 1
Figure 1. Dissection microscope images of particular aspects of neonatal mouse cortex and DRG isolation. (a) Dorsal view of a freshly extracted neonatal mouse brain. The dotted lines indicate the area where incisions must be made to facilitate the removal of the meningeal layer. (b) Ventral view of brain, dotted lines indicate the area where the cortex meets the ventral diencephalon. Deep incisions must be made along the dotted lines to aid the isolation of the cortices. (c-c') Visual depiction of how to pry the cortex away from the remainder of the brain. (d) A freshly isolated P5-P10 mouse spine prior to trimming away excess muscle and bone (d'). (e) Location of DRGs within the spinal column. (f) The approximate number of DRGs that should be isolated from one mouse. (g) A DRG with long roots that require trimming prior to enzymatic digestion. The dotted line indicates the region where the roots should be trimmed. (g') DRG post root-trimming.

Figure 2
Figure 2. OPCs are expanded within a mixed glial culture, purified, and subsequently differentiated as an OL-enriched culture. (a) Phase contrast images of mixed glial cultures at different stages of development. At DIV1, cells appear round with few flattened cells. Stratification of mixed glial culture begins at DIV3, where astrocytes form a uniform monolayer at the base of the flask, upon which OPCs proliferate. Many OPCs are seen at DIV8 (arrows) adhered to the surface of the astrocyte monolayer. (b) Once purified from the mixed glial culture, DIV1 OPCs have extended only a few processes. At DIV3, cells have extended many processes, reminiscent of intermediate-stage OLs. At DIV6, flattened OLs (asterisk) appear to have produced membranous sheets (dashed line). Scale bars, 50 μm.

Figure 3
Figure 3. Characterization of OL-enriched culture. (a) Confocal images of isolated OLs at different stages of development. NG2+ve OPCs have simple morphology, whereas MAG+ve OLs possess multiple arborous processes. MBP+ve OLs have extended membranous myelin-like sheets. Scale bar, 50 μm. (b) Purified OL-lineage cells originate as OPCs, and differentiate into MAG+ve, MBP+ve OLs over 6 DIV. At DIV1, all OPCs are NG2+ve, while none are MAG+ve or MBP+ve. At DIV3, MAG+ve and few MBP+ve OLs are now evident. The majority of OLs are MAG and MBP+ve at DIV6, with few remaining NG2+ve OPCs. Scale bar, 100 μm. (c) Mean values ± SD of the percent OL-lineage cells at different stages of development over 6 DIV. At DIV1, all OL-lineage cells are NG2+ve, accounting for 50±14% of total cells within the culture. At DIV3 and DIV6, OL-lineage cells respectively account for 36±6.8% and 32±8.4% of total cells, consisting of varying proportions of NG2+ve, MAG+ve and MBP+ve OLs. (d) SDS-PAGE performed on protein derived from enriched OL-cultures demonstrating the graded expression of OL-markers CNP and MBP over the 6 DIV culture period.

Figure 4
Figure 4. Characterization of the DRGN culture pre-OPC seeding. (a) Phase contrast images of DRGNs over the 9 DIV culture period pre-OPC seeding. DRGNs originate as large-bodied cells with few processes, and produce an increasingly complex neurite network. Scale bar, 100 μm. (b) Confocal images of DRGN cultures fixed at DIV9 (pre-OPC seeding) and stained for neuron-specific markers Tuj1 and NF200. DRGNs have produced a neurite network upon which OPCs may be seeded to produce OL/DRGN myelinating co-cultures. Scale bar, 50 μm.

Figure 5
Figure 5. OLs co-cultured with DRGNs result in OL-mediated wrapping of DRGN neurites with MBP+ve membrane. (a) A 4-field confocal image montage of a DIV6 OL/DRGN co-culture. Many MBP+ve OLs can be seen interacting with the underlying DRGN neurite bed. Scale bar, 100 μm. (b) A magnified confocal view of MBP+ve OLs wrapping multiple DRGN neurites. Scale bar, 50 μm. (c) Digital magnification of the region denoted in (b) where a DRGN neurite is being wrapped with OL membrane. Scale bar, 25 μm.

Discussion

This report describes a method for isolating murine OPCs for differentiation in OL-enriched cultures or OL/DRGN co-cultures. When cultured alone, the OPCs differentiate into MBP+ve OLs, producing myelin-like membranous sheets. When added to DRGN neurite beds, OLs enwrap the DRGN neurites with MBP+ve membrane. This model benefits the investigation of the complex underpinnings governing OL-mediated axonal ensheathment.

While of great value, the establishment of such cultures is technically challenging. In particular, demanding aspects include efficient tissue digestion/dissociation, maintenance of balanced culture media pH, and DRGN media changes. It is important to consider that the length of digestion, amount of tissue being digested and the amount of trituration affects the efficacy and end result of the tissue dissociation. It is not unusual for experienced researchers to obtain low cellular yields from dissociated nervous system tissues. In addition, murine OPCs tend to be sensitive to changes in the pH of the culture media, particularly under alkaline conditions. The maintenance of cultures at 8.5% CO2 aims to prevent this, since OPCs appear to better tolerate slightly acidic conditions over basic. With regards to feeding DRGNs, media changes must be performed quickly as to not desiccate the neurons, however, must be gentle as to not disrupt the developing neurite bed. Abrupt media changes may dislodge the neurite bed from the substrate, and likely result in its complete dissociation from the coverslip.

The potential merit of this model system greatly overshadows its technically demanding nature. One advantage of this system is the use of post-natal mice for cell culture derivation, circumventing the need to sacrifice breeding females to harvest embryonic tissue. Another advantage is the lack of requirement for growth factors (GFs) for the expansion of OPCs. Mixed glial cultures provide an environment that supports the propagation of OPCs, presumably due to the presence of astrocyte-derived trophic factors. Other methods, such as derivation via neurospheres7,8, rely on the mitogenic properties of GFs such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) for OPC expansion. Similarly, using postnatal (P5-10) mice for DRGNs avoids the requirement of supplementing the culture media with nerve growth factor (NGF), a neurotrophic factor required for the in vitro survival of embryonic DRGNs 9, 10. It is of interest to avoid using NGF as it negatively influences the myelinating capacity of OLs when cultured with DRGNs 4. Avoiding the use of GF-supplemented media also has economic benefits, as these reagents become costly when used on large scale.

Perhaps the most important benefit of this culture model is its derivation from mouse-only tissues, thus providing the opportunities to derive both OPCs and DRGNs from the wide variety of transgenic mouse lines. This allows for the study of both DRGN and/or OPC-specific properties that govern myelination. This will be especially important for elucidating the receptor/ligand interactions regulating OL-mediated myelination of axons. In all, this technique is of great value with regards to neuroscience research due to its applications towards understanding the molecular cues underlying myelination.

Disclosures

No conflicts of interest declared.

Acknowledgments

This project was funded by a grant from the Multiple Sclerosis Society of Canada to R.K. R.W.O is a recipient of a Studentship from the Multiple Sclerosis Society of Canada. S.D.R is a recipient of Post-Doctoral Fellowships from the Multiple Sclerosis Society of Canada and Canadian Institutes of Health Research.

Materials

Name Company Catalog Number Comments
Dulbecco's Modified Eagle Medium (DMEM) Multicell 319-005-CL
Hank's Balanced Salt Solution (HBSS) Invitrogen 14170-112
Minimum Essential Media (MEM) GIBCO, by Life Technologies 12360-038
Fetal Bovine Serum (FBS) GIBCO, by Life Technologies 10091-148
Penicillin-Streptomycin (Pen/Strep) GIBCO, by Life Technologies 15140-122
GlutaMAX Invitrogen 35050-061
Poly-l-lysine Sigma-Aldrich P2636
Bovine Serum Albumin (BSA) Sigma-Aldrich A4503
Human merosin purified protein (LN2) EMD Millipore CC085
Recombinant rat ciliary neurotrophic factor (CNTF) PeproTech Inc 450-50
L-thyroxine Biochemika 89430
Holo-transferrin Sigma-Aldrich T0665
B27 supplement GIBCO, by Life Technologies 0080085-SA
Bovine insulin Sigma-Aldrich I6634
3,3',5-Triiodo-L-thyronine Sigma-Aldrich I6634
Progesterone Sigma-Aldrich P8783
Putrescine Sigma-Aldrich P7505
Sodium Selenite Sigma-Aldrich S5261
5-Fluoro-2'-deoxyuridine (FuDR) Sigma-Aldrich F0503
Papain solution Worthington Biochemical LS003126
DNaseI Roche Group 1010159001
L-cysteine Sigma-Aldrich C7352
24-well tissue culture dishes Cellstar 662-160
T25-tissue culture flasks with vent cap Corning 430639
10 cm tissue culture dishes Corning 430167
10 cm petri dish Fisher Scientific 0875713
Collagenase A Roche Group 103578
CellTrics 50 μm filter (optional) PARTEC 04-004-2327
Myelin Basic Protein (MBP) Antibody AbD Serotec MCA409S
NG2 antibody EMD Millipore AB5320
Myelin-Associated Glycoprotein (MAG) antibody EMD Millipore MAB1567
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) antibody Covance SMI-91R-100
Actin pan Ab-5 antibody Fitzgerald 10R-A106AX
Neurofilament-200 (NF) antibody Sigma-Aldrich N4142
Tubulin beta-3 chain (Tuj1) antibody EMD Millipore MAB5544
Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody Invitrogen A11008
Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary antibody Invitrogen A21422
Alexa Fluor 647 goat anti-rat (IgG) (H+L) secondary antibody Invitrogen A21247
Goat anti-mouse IgG (H+L)-HRP conjugated secondary antibody Bio-Rad 170-6516
Goat anti-rat IgG (H+L)-HRP conjugated secondary antibody Santa Cruz Biotechnology, Inc. SC-2065
4',6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich D9542

Media Recipes

100X OL-Supplement*

IngredientAmount to add
DMEM100 mL
BSA1.02 g
Progesterone0.6 mg
Putrescine161 mg
Sodium Selenite0.05 mg
3,3',5-Triiodo-L-thyronine4 mg

*Store at -80 °C in 250 μL aliquots

OL media

IngredientAmount to add
DMEM23.75 mL
100X OL-Supplement250 μL
Bovine insulin (from 1 mg/mL stock)125 μL
GlutaMAX250 μL
Holo-transferrin (from 33 mg/mL stock)37.5 μL
B27 Supplement500 μL
FBS125 μL
CNTF (from 50 ng/μL stock)25 μL

Mixed glial culture media (made up in DMEM)

IngredientFinal concentration
FBS10%
Pen/Strep (0.33% from stock)33 units/mL Penicillin and 33 μg/mL Streptomycin
GlutaMAX1%

DRGN media (made up in DMEM)

IngredientFinal concentration
FBS10%
Pen/Strep (1% from stock)100 units/mL Penicillin and 100 μg/mL Streptomycin

Digestion Solution Recipes:

OPC papain solution (made up in MEM)

IngredientFinal concentration
Papain solution1.54 mg/mL
L-cysteine360 μg/mL
DNaseI60 μg/mL

DRG papain solution (made up in HBSS)

IngredientFinal concentration
Papain1.54 mg/mL
L-cysteine360 μg/mL

DRG Collagenase A solution (made up in HBSS)

IngredientFinal concentration
Collagenase A4 mg/mL

DOWNLOAD MATERIALS LIST

References

  1. Avellana-Adalid, V. Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential. J Neurosci Res. 45, 558-570 (1996).
  2. McCarthy, K. D., de Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85, 890-902 (1980).
  3. Barres, B. A. Cell death and control of cell survival in the oligodendrocyte lineage. Cell. 70, 31-46 (1992).
  4. Chan, J. R. NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes. Neuron. 43, 183-1891 (2004).
  5. Camara, J. Integrin-mediated axoglial interactions initiate myelination in the central nervous system. J Cell Biol. 185, 699-712 (2009).
  6. Ishibashi, T. Astrocytes promote myelination in response to electrical impulses. Neuron. 49, 823-832 (2006).
  7. Chen, Y. Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc. 2, 1044-1051 (2007).
  8. Pedraza, C. E. Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors. Glia. 56, 1339-1352 (2008).
  9. Lewin, G. R., Ritter, A. M., Mendell, L. M. On the role of nerve growth factor in the development of myelinated nociceptors. J Neurosci. 12, 1896-1905 (1992).
  10. Greene, L. A. Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons. Dev Biol. 58, 106-113 (1977).

Comments

46 Comments

  1. very nice video and presentation....

    Reply
    Posted by: Anonymous
    December 28, 2011 - 12:44 PM
  2. Nice explanation. I have been carrying out this procedure for some time now and find that it works quite well.

    How many OPCs you generally obtain from a single mouse brain?

    Reply
    Posted by: Anonymous
    January 26, 2012 - 4:35 PM
  3. Hi - thanks for your comment, typically I yield about ²00,000 OPCs per mouse brain (on average).

    Reply
    Posted by: Anonymous
    February 2, 2012 - 1:04 AM
  4. Excellent. I am using B6 mice, usually P4-5 (because we also take cerebellar neurons from these same pups). I usually yield between 100-130,000 cells/brain. I see some differences in our protocols (I seed three brains/T75, use trypsin instead of papain, etc.). What steps have you found to dramatically increase your OPC yield post shake-off?

    Reply
    Posted by: Anonymous
    February 2, 2012 - 12:17 PM
  5. The largest factor in OPC yield is the efficiency of your digestion (ie. minimal cell death). In addition, the age of the mice from which you isolate the mixed glial cultures is a factor. I think if you go too far past P², many OPCs will be postmitotic, and therefore limit their proliferation capacity. I usually try to go as young as possible (P0) as this seems to work best. If you absolutely need to use P4-P5, maybe try to culture your monolayers longer than 9 days - try waiting ² weeks prior to shake off. Hope this helps - RO

    Reply
    Posted by: Anonymous
    February 2, 2012 - 1:06 PM
  6. Thanks for the great info. That is interesting, as many of the protocols for the "DeVellis-method" use different enzymatic preparations (trypsin +/- EDTA, +/- DNAase, papain, etc.). I had basically adapted my rat OPC protocol for mice, but am realizing that they are just not the same. I will trip your papain method and let you know how things pan out. Also, is there a reason you use T²5 flasks instead of combining brains into a T75?

    Reply
    Posted by: Anonymous
    February 2, 2012 - 4:55 PM
  7. No there is no specific reason for using T²5, simply this is what I have always been using. I would imagine combining 3 mice into a T75 flask would roughly be the same thing. Good Luck!

    Posted by: Anonymous
    February 7, 2012 - 12:00 PM
  8. Hi Ryan, nice video and explanation. Do you do lectin panning to further get rid of microglia or do you find that a 30 min incubation at 37 degrees is enough? Also, how do you know cells are ready to shake? are they always ready at day 9? what do you look for specifically?
    thank you

    Reply
    Posted by: luisa t.
    April 10, 2013 - 12:39 PM
  9. hello...I have gone through this video...very nicely presented...but I have one doubt that if I put the flasks on shaker which only maintains temp and speed but not CO² level, the pH will change...would it not allow the OPCs to survive at all??? and can I use HEPES in medium to solve this problem???
    M willingly waiting for your reply....

    Reply
    Posted by: Anonymous
    February 4, 2012 - 12:35 PM
  10. Thank you for your comment, you are correct in that it will not work without CO² buffering. HEPES buffer will likely not solve this problem either. I'm afraid you will absolutely need to have CO² buffering for this isolation to work.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 11:56 AM
  11. Hi, very INFORMATIVE video, I zalak parikh working on glial cells. In your protocol you are not using any filter after papain digestion but I am facing one problem of Fibroblasts contamination. What could be the possible solution for this problem?
    thank you
    Zalak.

    Reply
    Posted by: Anonymous
    February 24, 2012 - 7:59 AM
  12. Hi Zalak, thanks for your comment. What glial cell type are you trying to culture? Oligodendrocytes, astrocytes or microglia? I have never experienced fibroblast contamination.. are you sure they are fibroblasts?

    Reply
    Posted by: Anonymous
    February 24, 2012 - 10:47 AM
  13. Hi there. Excellent video, very informative. I was just wondering if you can incubate the flasks in 5% CO² instead of 8% CO²?
    Thanks
    Matt

    Reply
    Posted by: Anonymous
    April 11, 2012 - 12:48 PM
  14. Hi Matt, Actually no, you need to have these at at least 8.5% CO² - otherwise you will have trouble establishing your mixed glial cultures - Good luck

    Reply
    Posted by: Anonymous
    April 11, 2012 - 10:22 PM
  15. Hi Ryan. Very nice video and presentation.
    I have to establish a DRG neuronal culture. Majority of all the papers I've read are using a embryonic source of DRG unlike you using post-natal mouse. What do you think will be the major difference between the cultures of the two??

    Reply
    Posted by: Lipi B.
    October 1, 2012 - 12:23 PM
  16. Hi Lipi,
    You're right most papers use embryonic DRGs. The benefit of using post-natal DRGs is that you don't have to sacrifice the mother, and if you use P5-P10 mice, there is no need to use nerve growth factor (in my hands). Embryonic DRG neurons require NGF supplementation to the media for their survival. In terms of the difference between cultures, embryonic dervied ones may have a bit more contaminating cells, since I find they divide pretty quickly. Otherwise I imagine they would be the same as post-natal derived cultures. Good luck!

    Reply
    Posted by: Ryan O.
    October 1, 2012 - 4:37 PM
  17. Thank you Ryan.

    Reply
    Posted by: Lipi B.
    October 3, 2012 - 2:39 AM
  18. Thank you Ryan.

    Reply
    Posted by: Lipi B.
    October 3, 2012 - 2:51 AM
  19. Great article Ryan. I had a question for you. Our lab generates mixed glial cell cultures by isolating NPCs from P1 mouse pup brains,expanding the NPCs via neurospheres in the presence of GFs, and then plating the spheres on matrigel for subsequent differentiation without GFs. My main goal in using the neurosphere protocol is to obtain OPCs, but I routinely get only ²0-30% O1+ OPCs following 8 days of differentiation on matrigel. GFAP+ astrocytes seem to dominate the whole culture, with OPCs growing on top of them. I wanted to try to incorporate your protocol into the neurosphere protocol by dissociating the spheres using TrypLE or Accutase, plating them on Poly-D-Lysine, letting the astrocytes grow to confluency while having OPCs proliferate on top, and then use the shaking technique to isolate OPCs. Do you think this is possible? When we perform in vitro studies with the NPCs, we usually plate the neurospheres on matrigel for a day and then trypsinize the cells for seeding into new dishes. Not only do I think the trypsin digest kills many OPCs, but I also remove astrocytes from the matrigel that then contaminate my cultures. Thanks for the help!

    Reply
    Posted by: Brett M.
    November 3, 2012 - 7:37 PM
  20. Hi Brett - To answer your question, yes I think it would be possible to do what you are suggesting. However, it seems to be a pretty roundabout approach. I think the more streamlined approach would be to simply seed your dissociated P1 neural cells onto PLL substrate to generate a monolayer. That way you would avoid having to generate neurospheres (which I imagine takes about 10 days) and then subsequently generating an astrocyte monolayer with the spheres (which would take another 10+ days). In my experience, OPCs don't like trypsin. If you wanted, you could try 0.05% Trypsin instead of 0.²5% and that seems to be more gentle. And if you don't incubate for too long, most astrocytes should remain on the plate. Hope this helps! -Ryan

    Reply
    Posted by: Ryan O.
    November 5, 2012 - 12:14 PM
  21. Ryan, thanks for the help! I tried your protocol and everything appeared to go well. However, one issue I ran into occurred during the overnight shaking step and subsequent differential adhesion assay to isolate a more pure population of OPCs. What I noticed was that after the overnight shaking I had many large oligospheres (maybe 10-²0 cells in size). These spheres did not stick down during the 30 minute plate incubation and I had difficulties trying to dissociate them as you described using the P1000 and P²00 tips. When I took the cells counts and plated the cells onto chamber slides, many smaller clumps remained and stuck down to the matrix. However, the clumps did not flatten out on the gel matrix very well (like neurospheres do) and eventually flaked off after about 5 days of cultures, leaving only sparse population of OPCs. Have you ever encountered this problem? I was thinking of seeding at a higher cells density than what you describe in the protocol.

    Reply
    Posted by: Brett M.
    December 2, 2012 - 10:21 PM
  22. Hi Brett - sounds like you're doing everything right. Normally I see lots of these "oligospheres" as a result of the overnight shaking protocol. However, I find that these are not the majority of OPCs, as there are many single cells and cells in clumps of ²-4. The oligospheres are difficult to dissociate, and due to this, I generally do not go out of my way to try and break them up. In my hands, these spheres spread out very nicely after about ²4hrs on Laminin-² substrate. You can try to seed at higher densities if you like, but the seeding density should be determined based on the experiment you want to conduct. For example, if you want to perform immunocytochemistry, it may not be ideal to have a very dense cell culture. Also, it's possible the clumps aren't super compatible with your matrix substrate - I always use Laminin. I hope this helps :)

    Reply
    Posted by: Ryan O.
    December 3, 2012 - 11:42 AM
  23. HI Ryan, very informative and nice presentation. I have tried your protocol and everything seem to work fine except at the overnight shaking. There were very few OPCs isolated and it seems there are still many OPCs adhere to the astrocytes. Have you encounter this issue?

    Reply
    Posted by: Esther W.
    December 13, 2012 - 3:25 AM
  24. Hi Esther, Thanks for your comment. How many OPCs are you yielding per mouse? I usually yield on average ²00,000 OPCs per mouse brain, but this is quite variable. At times I have yielded 500,000 or 50,000 per brain, and I'm not sure what accounts for this variability. In any case, you will never dissociate 100% of the OPCs from the monolayer, I've noticed a lot remain stuck. My advice would be to simply give it another shot and see if your yields are a bit higher next time. Hope this helps - RO

    Reply
    Posted by: Ryan O.
    December 16, 2012 - 5:35 PM
  25. Thanks Ryan for your advice. My yield is definitely much lower than yours. I will definitely try another round. :)

    Reply
    Posted by: Esther W.
    December 16, 2012 - 8:24 PM
  26. HI Ryan, I face the problem of getting immature OPC instead of mature OPC after 6 days of OL-enriched culture. What is your view?

    Reply
    Posted by: Esther W.
    January 23, 2013 - 2:51 AM
  27. Hi Esther, you should always expect to have some OPCs that don't differentiate during the timecourse. However, the majority of OLs should at least be expressing MAG, if not MBP at DIV6. My guess would be that your media is not exactly correct. Make sure that all the ingredients are at the correct concentration in your 100x OL-supplement stock - especially tri-iodothyronine and L-thyroxine (4mg per 100mL). I hope this helps, let me know if you still have problems.

    Posted by: Ryan O.
    January 23, 2013 - 12:56 PM
  28. Thanks Ryan for replying. I have added tri-iodothyronine only as there is no mentioned of L-thyronine in the 100x OL-supp recipe. Can i still add L-thyronine when the OL-enriched is on DIV5?

    Posted by: Esther W.
    January 23, 2013 - 8:05 PM
  29. Thanks for notifying me of this mistake, I'll notify the editorial team ASAP. Yes you can try to add it on DIV5, but no guarantees this will drive them any farther since they are aleady 5 days in. Give the protocol a second try, and let me know if the lack of differentiation still occurs. -Ryan

    Reply
    Posted by: Ryan O.
    January 24, 2013 - 12:47 AM
  30. Hi Luisa thanks for your comment :) I don't do lectin panning, as I find that incubation on the culture plates is enough to get rid of most of them, but if you're having a problem with microglia that is an additional option. I usually shake on day 9, but I've found you can shake anywhere from 9 to 1² days, but any longer than that you will get less OPCs. Generally if you see a lot of cells on top of the astrocyte monolayer, they are ready to go. However in my experience this dŒsn't really happen before day 9. Hope this helps RO

    Reply
    Posted by: Ryan O.
    April 10, 2013 - 2:28 PM
  31. Hi Ryan.Thanks for this protocol. I did exactly what you have described in this protocol, except the shaking step. I did it without CO2, but the caps are tightly sealed, how is it possible that CO2 will effect pH of tightly sealed flask? My yield was very low, besides I had fibroblast contamination as well. I used uncoated flasks as well, because once during the shaking all the layer came off the bottom of the flask, what do you think would be the reason for this?.And in my experience when FBS is used in OPC culture media it made OPC's to differentiate to astrocytes rather then oligodendrocytes.Overall I couldnt replicate this procedure unfortunately and trying to find other methods..

    Reply
    Posted by: Aysel J.
    February 20, 2014 - 5:33 PM
  32. Hi Aysel - from what you describe in your message, you didn't follow my protocol.

    1) So you didn't shake the flasks? Or you didn't use the same method of shaking?

    2) As far as I know, all standard tissue culture incubators have some level of CO2 buffering (usually 5%). Strictly speaking, if you tighten the cap of your flasks, there will be no gas exchange between the incubator and the flask, if you are using plugged caps. I use vent-capped flasks (0.22um pore), so gas exchange occurs between the flask and the incubator when the caps are tight. To answer your question, CO2 buffering will have little-to-no effect on the pH of the media if you are using tightly-screwed plugged flask caps.

    3) I haven't experienced fibroblast contamination in my cultures, I imagine this would be a dissection issue. The only source of fibroblasts I can imagine would be from the meninges, so be sure to fully remove this prior to dissociation of the cortex. Unless you are confusing fibroblasts for astrocytes. Too many astrocytes is a result of too much agitation at the differential adhesion step.

    4) I have always used coated flasks, in my experience OPCs will not adhere to uncoated surfaces. I am willing to bet you'll get almost no OPCs if you continue to use uncoated flasks. If you want you can try to use other substrates such as PDL, poly-O-ornithine, collagen etc.

    5) I haven't noticed any significant transformation of OPCs into astrocytes in the presence of FBS. I imagine this might occur more frequently in immortalized OPC cell lines. Perhaps that's what you are thinking of.

    I hope this helps, otherwise best of luck in your research.

    Reply
    Posted by: Ryan O.
    February 20, 2014 - 8:52 PM
  33. Hi Ryan, thank you ver much for detailed answer. Sorry for my english, i couldnt explain good.
    I did the shaking of course but without putting shaker inside the CO2 incubator, I did shaking at 37 degree incubation only and at 180 rpm. Because the flasks are uncoated(I have used uncoated flasks for rat OPC"s without problem, that is why I tried this way with mouse as well) I hesitated to increase rpm to 240 or so, to prevent astrocyte detachment, and I also think that fibroblast contamination is because of the meninges. Second time I will prevent it.
    It is good to know that I must use coated flasks, I will try again, if you say so.
    Thank you so much, good luck to too..

    Reply
    Posted by: Aysel J.
    February 21, 2014 - 6:22 AM
  34. Hi Again,
    Alright - I strongly suggest to put the shaker in the incubator for the overnight shake, as I have found this to be a crucial step in maintaining pH balance. The media on your cells should always be a rich red colour (indicative of a balanced pH) all the time. Mouse OPCs are very sensitive to pH, and will die with too much fluctuation. If during your shaking, the media becomes at all purple-ish or yellow-ish, your OPC yield will drastically reduce. I wouldn't be surprised either if this was a contributing factor to the monolayer detachment you experienced previously. There is a significant difference in the in vitro behaviour/viability of mouse OPCs as compared to rat, hence the generation of this protocol. Your best bet is to simply follow this protocol exactly as is.

    Thanks for your comment, and good luck,
    Ryan

    Reply
    Posted by: Ryan O.
    February 21, 2014 - 12:25 PM
  35. Hi.
    One more question Ryan, about amount of the brains seeded, I was seeding 2 rat brains/ T75 flask , in the mouse case to obtain same amount of cells seeded I was using 6 brains per flask. Would you suggest to use 3-4 instead of six? and if i seed 6 animals should I increase concentration of insulin added to mixed culture media? Overall what would be optimum amount of brains per T75 flask in your opinion? Does it matter to much? Because here you use 1 brainper T25 flask.
    Thanks again..

    Reply
    Posted by: Aysel J.
    February 26, 2014 - 4:20 PM
  36. Hi Aysel,
    Again, your best bet for success is to follow the protocol exactly as is - T25 flasks are what this method has been optimized for. I suppose T75 flasks have 3 times the surface area, so in theory 3 mice would have to be seeded to a T75 to yield the same cell density. I've never tried this protocol with T75 flasks, and I know that different culture vessels have different impact on growth and survival of OLs.
    Best - RO

    Reply
    Posted by: Ryan O.
    February 27, 2014 - 10:47 AM
  37. Hi Ryan . I want to say that I replicated the procedure exactly as here, and have got very good yield, but I have 1 more question to you, majority of OPCs die in the differentiation media after 6-7 days of incubation. You indicated that it is important not to distrub them until fixation, but how do you do the medium chage, I put 1 ml media first day and did 2/3 media change each 3 days, I seeded all cells to one 24 well format plate, at each time points(3 d, 6 d, 9 d) I transfer the slides to new plate and fix them there and do medium change for rest of the slides. By the way I culture OPCs at 5% CO2, becaue I have only 2 options 5% and 10%, i cant adjust ph of the incubators at this time point. Is it the contributing factor for death of the cells after 4-5 days of incubation?

    Reply
    Posted by: Aysel J.
    March 19, 2014 - 2:43 PM
  38. Hi Aysel - I'm glad to hear things are looking up -

    1. I don't suggest differentiating the OLs for longer than 6 days. If you're finding they are not differentiating fast enough, you can add 4 mg of L-Thyroxine to the 100X OL-supplement (see the media recipe list above).

    2. It is normal to lose OLs over the time course of differentiation. That being said, you don't want to encourage death by moving the cells or culturing at low pH. You should not be changing the media of the purified OL cultures at any point - they do not require media changes. The protocol also says to culture the OLs at 8.5% CO2. Since you have access to both 5 and 10% CO2 incubators, and 8.5 is closer to 10 than it is to 5, I suggest culturing them at 10%.

    3. Rather than culturing all of your time points in one dish, use 3 dishes (one for 3 days, one for 6 etc). That way, you won't have to disturb your other time points.

    4. Do not remove the coverslips from the wells of your 24 well dish during fixation - simply add concentrated PFA directly into the media and allow to fix for about 15 mins at room temp.

    Sounds like you're almost there, Best of luck - Ryan

    Reply
    Posted by: Ryan O.
    March 19, 2014 - 3:58 PM
  39. Let me write it again to see if I have understood you correct, you say dont do any media change ever, neither at 3 days or 6 days or further? The other things I will try what you suggest, culturing and seeding in different plates at 10% CO2
    I want to thank you a lot for your help and all of the explanations.
    Thanks ..
    Best,
    Aysel

    Reply
    Posted by: Aysel J.
    March 19, 2014 - 4:22 PM
  40. Thats correct :)
    You're very welcome,

    Ryan

    Posted by: Ryan O.
    March 19, 2014 - 5:49 PM
  41. Hi Ryan,

    Thanks for sharing this protocol, looks really good and will try it out. I have a couple of questions regarding your protocol and was hoping you could shed some light on some problem I am experiencing at the moment.

    With regards to what you have just described, we actually perform a very similar mixed glial culture on P0-P3 rat brains. Our isolation stages are very similar with the exception we use DNAse1 and L-cysteine along with Papain. Upon plating our cell suspensions the flasks are densely populated with microglia on top, then opc's below and then astrocytes. We basically shake off our microglia before doing the overnight shake for the OPC's.

    My question is how do you get rid/ not see any microglia in your mouse cultures? Is it also possible to obtain microglia from your method?

    We are currently experiencing problems with obtaining OPC's from our overnight shake (in rat). The cells look healthy when in culture with the microglia but once the microglia are removed and we perform the steps required to plate the OPC's we have a lot of contaminants in with the cells, almost like long fibrous structures that are stained by trypan blue. We still obtain + 5 million OPC's but the cells do not look healthy and the morphology is not consistent with OPC's. Upon plating these cells they fail to show characteristics of OPCs and a lot die off. I will mention we do not shake in an incubator but in an orbital shaker at 250rpm with the caps sealed tight and parafilm. Have you ever encountered this?

    Many thanks .

    Reply
    Posted by: Graeme I.
    February 9, 2015 - 1:06 PM
  42. Hi Graeme, thanks for your question.
    We do in fact get microglia in our mixed cultures, but we remove them prior to the overnight shake with a slow short shake (as in the rat method). These shaken off microglia are a very pure population, and can be cultured separately if desired.
    With regard to the problems you’re encountering shaking your rat cultures, I can’t say I’ve experienced anything quite like what you’re describing. It almost sounds like your cultures are suffering from contamination (maybe yeast?). We’ve experienced contamination a couple of times, and it resulted in much of the monolayer dissociating from the base of the flask during the overnight shake. If you like, you can send me a pic of the fibrous structures in the hopes that I could recognize what they are (romea077@uottawa.ca).
    We have never had luck shaking flasks in an orbital shaker – we exclusively use tissue culture incubators with vented caps. One risk of using an orbital shaker is they pose a threat for contamination, as they are often used for bacterial cultures.
    I hope this helps and best of luck,
    Ryan

    Reply
    Posted by: Anonymous
    February 20, 2015 - 11:12 AM
  43. Hi Ryan,

    Thanks for your reply.

    I tried the protocol and achieved very nice OPC's so everything seems ok there.

    With regards to what I was seeing after my rat OPC shake, I believe it not to be of any contamination but more something coming out of solution in our media. As a test I shook DMEM10% in a T75 flask, 37 degrees, 250rpm overnight. The next day I took an aliquot and looked at it with trypan blue. To my surprise I saw the same fibrous clumps that I was seeing after the OPC shake

    . My DMEM only contains FBS and pen-step so this has to be something in FBS. My most recent shake off I decided to shake off 5 flasks with DMEM 10% and 5 flasks with OPC media (our OPC media contains 2.5ml FBS vs 50ml in DMEM). The next day after following normal protocol I counted the cells from both conditions. The OPC media flasks had almost none of the fibrous clumps whereas the DMEM flasks had alot. I can now partially get round this problem by filtering with 40uM strainer but have you ever heard or seen this kind of thing. We have used several batches if FBS so it cannot be that every batch has precipitated proteins in it but perhaps what were doing is causing precipitation of FBS proteins that are affecting the OPC's?

    Thanks

    Graeme

    Reply
    Posted by: Graeme I.
    March 15, 2015 - 4:16 PM
  44. Hi Ryan,

    Is your FBS from Invitrogen decomplemented ? If so did you buy decomplemented or did you heat it at 56*C and for how long.

    Thanks

    Reply
    Posted by: Ludovic D.
    February 24, 2017 - 10:40 AM
  45. Hi Ludovic,
    I've used FBS that has been heat-inactivated by the manufacturer, and I've also used FBS that I've heat inactivated myself. For the latter, if memory serves, about 30 minutes at roughly 50 degrees C should do the trick (water bath).
    Good luck, Ryan

    Reply
    Posted by: Anonymous
    February 26, 2017 - 11:49 AM
  46. Hi Ryan,
    I am wondering if you have any experience with adding Cre Recombinase Adenovirus to the cultures? And if you think a particular day would be best to add it? I was thinking of adding it on D8 (1 day before the full media change) but wasn't sure if adding virus and then shaking the cells would cause too much stress?
    Thanks,
    Megan

    Reply
    Posted by: Megan R.
    April 30, 2018 - 2:39 PM

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