Department of Neurobiology and Behavior, University of California, Irvine
Rahman, A. S., Parvinjah, S., Hanna, M. A., Helguera, P. R., Busciglio, J. Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures. J. Vis. Exp. (45), e2384, doi:10.3791/2384 (2010).
In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.
1. Cryopreservation of Cortical Tissue Blocks
Cleaning, Chopping, and Freezing
2. Thawing and Culturing of Frozen Cortical Tissue Blocks
Note: Media names appended with crosses (+) indicate the inclusion of additives to the base composition of the media. In this text, DMEM(++) denotes DMEM plus 10% EBS plus 1% AA, while NB(+++) denotes NB plus 1% AA plus B27 plus N2.
Thawing and Culturing
3. Representative Results
Healthy cultures will show significant growth and differentiation after 5 days post-thawing and will usually stabilize in its growth by 10 days (Figure 1). Immuncocytochemical staining of these cultures reveals numerous astrocytes (Figure 2A) and neurons (Figure 2B) for both human and rat primary neuronal cultures. This protocol is also suitable for the generation of NPCs as free-floating neurospheres (Figure 3A), which under differentiating conditions, result in high quality mixed cultures (Figure 3B,C).
Figure 1. Cell cultures of frozen human cortical tissue. Frozen human cortical tissue blocks are thawed and plated on poly-lysine coated coverslips and grown for 10 days. By day 5 cell expansion is apparent and by day 10 confluency is achieved. Scale bar: 100 μm.
Figure 2. Immunocytochemical staining of cell cultures. (A) Cultures were stained with the glial marker glial fibrillary acidic protein (GFAP, thick arrows). (B) Neurons were detected with the neuronal marker beta-tubulin III (TIII, thin arrows). (C) Both human and rat cultures show abundant glia and neurons after 10 days in culture. Primary antibodies: mouse anti-GFAP (1:1000) and rabbit anti-TIII (1:1000). Secondary antibodies: anti-mouse Alexa 594 (1:500) and anti-rabbit Alexa 488 (1:500). Nuclei staining: Hoestch blue (1:1000). Scale bar: 50 μm.
Figure 3. Generation of neurospheres. (A) Frozen tissue was processed as described above and allowed to propagate as neurospheres. (B) Neurospheres were plated on glass coverslips under differentiating conditions and grown for 10 days, resulting in cells migrating away from the neurosphere. (C) Both neurons and astrocytes are present in the expanding fringe of a differentiating neurosphere. Nuclear counterstaining, primary and secondary antibodies used as in Fig 2.
Cryopreservation offers an opportunity to bank precious brain tissue samples for future use. Here we describe a simple but effective protocol to generate both neuron-enriched cultures and neuronal precursor cells from frozen brain tissue blocks. This economical procedure avoids the costs of traditional cryopreservation techniques that utilize more expensive rate-controlled freezers. The protocol allows for the generation of viable neuronal cultures post-thawing by providing a rapid yet effective means to freeze tissue blocks. The entire freezing process can take as little as 20 minutes. In addition to primary neuronal cultures, using this method tissue blocks can also be thawed to generate NPCs grown as free floating neurospheres. In this regard, the lack of serum in our freezing medium ensures that cells are preserved in an undifferentiated state. Under differentiating conditions, neurospheres produced from frozen tissue show expansion and differentiation rates very comparable to neurospheres generated from fresh tissue.
No conflicts of interest declared.
This research was supported by grants from the Undergraduate Research Opportunities Program at UCI (A.R. and S.P.) and grants from the State of California Alzheimer's Disease Initiative and the National Institutes of Health grant no. HD38466, and Alzheimer's Disease Research Center grant no. AG16573 (J.B.)
|Dulbecco’s Modified Eagle Medium (DMEM)||Invitrogen||11995-073||High gluclose 1X|
|Bovine calf serum supplemented||Hyclone||SH30072.03||For culture medium|
|Neurobasal-A Medium (1X), liquid||Invitrogen||1088-022|
|B27 Supplement||Invitrogen||17504-044||Supplement for Neurobasal medium|
|N2 Supplement||Invitrogen||17502-048||Supplement for Neurobasal medium|
|Trypsin (10X)||Cellgro||25-054CI||Tissue dissociation|
|Deoxyribonuclease 1 from bovine pancreas (DNase)||Sigma-Aldrich||D4527-30KU||Tissue dissociation|
|Hanks Balanced Salt Solution (HBSS) (10X)||GIBCO, by Life Technologies||14065||Cleaning tissue, washes, and freezing medium|
|Poly-L-Lysine- 500mg||Invitrogen||P2636||Substrate for adhesion of neuronal cells|
|antibiotic-antimycotic (100X), liquid||Invitrogen||15240-062||To prevent contamination|
|Large orifice pipette Tips (1-200 ul)||Fisher Scientific||02-681-141||To prevent shear stress to cells|
|Graduated pipette tips (101-1000 ul)||USA Scientific, Inc.||1111-2721|
|21 G1 precision guide needles||BD Biosciences||305165||To clean tissue|
|10 ml pipette||USA Scientific, Inc.||1071-0810||Individually wrapped|
|50 ml tubes||USA Scientific, Inc.||926-9-04|
|Single edge razor blade||Smith & Nephew Inc.||67-0238||To chop tissue|
|60 x 15 mm polystyrene petri dish||USA Scientific, Inc.||8609-0160||For general culture|
|100 x 15 mm polystyrene petri dish||USA Scientific, Inc.||8609-0010||For cleaning tissue|
|Cryogenic box||Nalge Nunc international||5026-1010|
|Freezing container||Nalge Nunc international||5100-0001||"Mr. Frosty"|
|2.0 ml cryogenic vials||Nalge Nunc international||5012-0020|
|DMSO||Fisher Scientific||D128-500||For freezing medium|
|Ethanol 200 proof||Sigma-Aldrich||E7023||For sterilizing razor blade|