We have successfully derived living stem and progenitor cells from human brain tissue that was frozen according to our protocol. This was achieved in virtually all samples. Since our initial testing of this procedure, more than 120 biopsy specimens have been stored this way. Tissue sources have been various grades of tumour (astroctoma, glioma, glioblastoma) and normal brain tissue described as SVZ, GM, WM, Cortex and hippocampus. It has been the policy of our lab to culture cells from all possible human tissue samples removed by surgeons of OUS’ Neurosurgery Department.

By three weeks a tissue fragment initially 8 cubic mm, cryogenically processed and then recovered many weeks later produced the same number of cells (about a million) as the number of viable cells initially retrieved from a fresh piece of the same tissue.

In the case of adherent cell cultures derived from normal brain tissue, the cell populations were comparable with those described by Murrell and co-workers for freshly grown cultures9. That is, they appeared to consist of neural stem cells and partially committed progenitors as described therein (Fig. 4a). There was one notable difference when comparing frozen-derived to fresh grown cultures: There was a statistically significant increase in the number of GFAP-positive cells. Since the proportions of cells in the cultures that were labeled with neuronal and oligodendrocyte markers were not different between the two sources of culture derivation it follows that more cells must be double labeled with GFAP. This implies that the process of tissue freezing and following revival of cells has somehow favoured an increased number of early progenitors that express GFAP. Double labeling of cells for the different neural sub-types in brain-derived cultures has been clearly shown to occur9. The contention that the progenitors derived from frozen tissue are more primitive is based on the currently held paradigm that as cells become more differentiated they become more restricted in their phenotypic repertoire. This issue nevertheless should be clarified in future studies.

POU5F1 assessment has been questioned recently as a reliable marker when using antibody detection13. We used one antibody for ICC, R&D clone 1759 which used human Oct 4 amino acids (aas) 1 to 265 as the immunogen. As well we did Western Blot9 with Cell Signaling mAb 4286. Known isoforms of Oct 4 protein range from 256 to 360 aas. Our Western Blot had one clear band at 46 kDa (the correct size) see9. Anyway results were consistent with those published in9.

The normal procedure worldwide for handling tumour material post-operatively has been to fix and freeze material for pathology assessment. These methods and storage protocols have precluded the possibility of obtaining living cells for analysis. Assessment of protein and RNA expression from these stored frozen samples has been possible but this was the extent of analyses. In terms of attempting to derive treatments some groups have used this dead tissue as a source of protein lysate to instruct dendritic cells for injection of autologous dendritic cell vaccines (DCVax14). Our lab has collaborated in the derivation of mRNA instructed DC vaccines where the mRNA has been derived from living tumour stem cells4. As well we have used these living TSCs to provide starting material for microarray analyses that compared TSCs to NSCs resulting in identification of potentially useful therapeutic targets in GBM5,15. To undertake studies of these TSCs’ and NSCs’ biology the availability of living cells has been an exciting opportunity. We have been able to undertake gene modifications enabling target gene knockdowns, followed by in vitro and in vivo assessment of tumorigenicity6,7,5. Whilst these exciting experimental opportunities have been enabled by our policy of culturing everything, it is often only later that we receive confirmation of diagnosis. With our new procedure enabling the storage of tissue that will yield live stem cells later, we can decide retrospectively which samples to study.

As well though there are economic incentives for following our freezing of viable tissue. Based on past experience it costs about four times as much (405 USD) for culture to three passages of living cells compared to 108 USD for preservation of tissue with our protocol. There are clearly difficulties in attending to biopsies out of hours, having staff availability, maintaining stored reagents etc. Our successful resurrection of live stem cells has alleviated many such concerns.

Because we have had the opportunity to use live cell cultures in our studies we have been able to use microarray and genomic comparisons between normal and tumour stem cells5,9,15 in order to identify possible gene targets. Following this, we have conducted experiments knocking down such targets with lentiviral vectors transduced into stem cells which have enabled assessment of gene function, relevance to tumorigenicity and so on5,6,7.

It has been a tradition to grow neural and brain tumour stem cells using the neurosphere serum free method first established by Reynolds and Weiss16 which we emulate in our VML medium. However this medium only produces successful cultures from 80% of glioblastoma samples and is very inefficient and unreliable for culturing normal human brain stem cells9. We have modified this medium to contain 1% serum and replaced EGF by TGFα. This medium we call Failsafe because it is indeed failsafe. All tumour samples (even low grade) and all normal brain tissue samples yield successful cultures using this medium. The serum used can be that of the patient (autologous) if patient treatment is to be derived from cultures. Cultures can be put from one medium to the other9. Sphere cultures can be grown adherently; adherent cultures can be grown as spheres9. Adherently grown cultures are tumorigenic in most cases when transplanted to the brains of SCID mice17. We hypothesized that small pieces of tissue could be stored in suitable cryo-preservative and later be used as a source to recover live stem cells. Having demonstrated this successfully we now have the capability to freeze tissue fragments first and later initiate the sorts of experiments that we can design at leisure.

The issues of growing and establishing stem cell culturing are diverse and complex. There are many confounding factors that need assessment to authenticate experimental results. Bona fide stemness needs to be regularly demonstrated. Minimum criteria for this is capacity to be cloned and multipotency9. As well the issue of phenotype assessment and reliability of methods needs clarity. Specially designed primers for qPCR may resolve some confusion but the occurrence of pseudogene sequences in DNA make this difficult. Western blot using monoclonal antibodies may well be necessary to establish expression of genuine protein markers. Alternative assessments such as facilitated cell sorting and analysis may well isolate subpopulations of cells with differing potential. In the case of tumour stem cells, the potential to demonstrate susceptibility to designer drugs or to supply gene information for the construction of personalized dendritic cell-based vaccines provide good motivation for these efforts. The same can be said for tissue-derived autologous stem cells and their potential for tissue regeneration technology. These future prospects make the protocol published here even more relevant and exciting as it gives greater scope and opportunity to think through well designed experiments without the pressure to instantly culture patients’ cells.