The role of cryopreservation techniques in manufacturing, transport, and storage of Car-T therapy products.

Several clinical trials have proved the efficacy and safety of T-cells chimeric antigen receptor (CAR-T cells) in treatment of malignant lymphoma and the first products were registered in the European Union in 2018. The shelf-life of CAR-T cell products in the liquid state is short, so cryopreservation offers a significant benefit for logistics in manufacturing and patient management. Direct shipment of the cryopreserved CAR-T cell therapy products to the clinical department is feasible, nevertheless, intermediate storage in the hospital cryostorage facility gives significant advantage in planning of their administration to patients. Moreover, some manufacturers prefer transport of the starting material cryopreserved at the collection site. The cryopreservation protocol used for starting material by the authors is based on combining dimethyl sulphoxide (DMSO) with hydroxyethyl starch (HES) and slow controlled cooling in cryobags housed in metal cassettes. This achieves the mononuclear cell post-thaw viability of 98.8 ± 0.5 % and recovery of 72.8, ± 10.2 %. Transport of the starting material to the manufactures and return transport of the CAR-T therapy product is performed by authorized courier companies. Intermediate cryostorage of the final CAR-T cell therapy product is performed in a separate dry-storage liquid nitrogen container. On the day of infusion, the cryopreserved products are transported to the clinical department in a dry shipper. On the wards the product is removed from the cassette, inserted into a sterile plastic bag, thawed in a 37 degree C water bath followed by immediate intravenous administration. The authors discuss the adherence of the used technology to good manufacturing practice (GMP) principles and genetic safety assurance rules. Doi: 10.54680/fr23310110112.

Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Standardisation of cell lines.

Standardisation of cell-based assays is significantly enhanced through the preparation and quality control of master and working cell banks. Validation of the cell substrate for use in an assay must address not only the assay conditions but also aspects such as culture passage level, pre-assay preparation and seeding of cells and pre-use validation of "critical reagents".

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells.

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. "mono-", "bi-" and "tri-cistronic" vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in antitumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation-time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

Quality assurance for cell substrates.

Animal cell lines are increasingly used in the manufacture of viral vaccines. They may play a key role in the preparation of seed stock virus and vaccine production. However, the same animal cell substrates may also be used for diagnosis of viral infection and surveillance of prevalent virus strains. Quality control of cell lines intended for use in this range of procedures is vital to ensure the absence of contaminating organisms and correct identity of the substrate cells used. Furthermore, the qualification and validation of the procedures, facilities and staff involved in the cell culture and testing are also important issues addressed in regulatory guidelines and regulations. The standards to which these activities are performed are dependent on whether the cells are intended for diagnostic and surveillance work or for seed stock virus isolation and production. This paper indicates when and how some of the relevant quality standards and quality control issues apply to cell lines intended for the different procedures involved in virus isolation and vaccine production.

Standardization in animal cell technology.

Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells. However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks. These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents. In addition to quality control techniques, culture conditions and growth medium used often require standardization. In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the 'performance' of cell lines. To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources. In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing. Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells.

New cell substrates for in vitro evaluation of microcystin hepato-cytotoxicity.

Algal toxins, as Microcystins released in water supplies, may represent a serious health hazard. The behaviour of primary hepatocytes was compared with that of immortalized liver cells, with the intention of providing a new test on Microcystin cellular toxicity. Immortalized liver cells were obtained by transfection with SV40 Large T antigen-bearing plasmids. Primary hepatocytes were used as a reference. Microcystin-LR at 10(-6), 10(-7)and 10(-9)m was added to hepatocytes maintained in suspension or cultured as three-dimensional hepatospheroids for 20 and 90 min at 37 degrees C. Toxic effects were monitored by cytoskeletal disruption ('blebs') using both light and scanning electron microscopy (SEM), lactate dehydrogenase release (LDH) and trypan blue dye exclusion test. Microcystin-LR at all doses induced bleb formation and a loss of microvilli in both primary hepatocytes and immortalized cell suspensions in comparison with controls. A high level of blebbed cells was detected in the absence of increased LDH release. The blebbing phenomenon was readily detectable by light microscopy but its morpho-complexity was unmasked by SEM, with early toxic events being indentified as loss of microvilli prior to bleb formation. Cells of primary hepatospheroids appeared to be less tightly attached to each other and more likely to bleb than immortalized ones. These immortalized cells could limit the use of primary cells and increase the reproducibility of the assay.

Hybridoma cell cultures continuously undergo apoptosis and reveal a novel 100 bp DNA fragment.

This report represents an investigation into the nature of apoptosis in hybridoma cultures and its significance to their utilization in biotechnology. To this end DNA fragmentation and capillary electrophoresis of genomic DNA was studied during the culture of two hybridoma cell lines. This indicated that the phenomenon of apoptosis was always present even under normal culture conditions. Two DNA fragments not associated with the typical DNA fragmentation ladder were identified in the two hybridoma cultures: a previously unreported DNA fragment of about 100 bp and a large fragment which may correspond to one reported in the literature (Walker et al., 1993). The small fragment was identified as soon as the early exponential growth phase of culture, while the large fragment appeared only in the latter part of the growth curve when there was marked DNA fragmentation. In addition we present evidence that aurintricarboxylic acid, which inhibits apoptosis in neural cells, permits this process in hybridoma cells at levels below 100 microM. This unusual predisposition of hybridoma cultures to undergo apoptosis and their response to inhibitor of apoptosis may have important implications for approaches to the culture of hybridomas and their utilization for monoclonal production.

DNA fingerprinting transforms the art of cell authentication.

The increasing diversity of new cell cultures is seriously stretching the capabilities of traditional methods of identification. DNA fingerprinting is set to play an important role in increasing confidence in the authenticity of cultures in research and industry.

Multilocus DNA fingerprint analysis of cell banks: stability studies and culture identification in human B-lymphoblastoid and mammalian cell lines.

The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.

The quality control of cell banks using DNA fingerprinting.

Reproducibility in animal cell culture technology requires careful preparation and characterisation of banks of cell cultures. The two standard techniques used in the quality control of such banks are isoenzyme analysis and cytogenetics which require complex and time-consuming procedures to enable cell line identification. However, DNA fingerprinting is potentially a more powerful method of analysis which can detect mutation and intra-species cross-contamination. At the European Collection of Animal Cell Cultures (ECACC) multilocus fingerprint analysis using probes 33.6 and 33.15 has been assessed in the quality control of cell banks. This method has confirmed consistency between master and working banks, has proven useful over a wide species range and can differentiate closely related cell lines. The key advantage of this method is its ability to detect cross-contamination by cell lines from a wide range of species using a straightforward and economical test. In addition the reproducibility of DNA fingerprints indicates their possible role in cell line authentication procedures which are important for patent and product licence applications.

Clonality of T cell and phenotypically undefined lymphoid neoplasms: the value of genotypic analyses.

The value of genotypic analysis for routine assessment of leukaemia and lymphoma was shown by the findings in a selected series of 30 cases. T cell receptor (TcR) gene rearrangements were observed in six out of nine cases of CD3+ CD8+ lymphocytoses and provided clear evidence for clonality in this group. The T cell proliferations in two of the remaining cases masked B cell lymphocytic leukaemia and hairy cell leukaemia, while in the third case no cause was found for the polyclonal proliferation. Heterogeneity of phenotype and genotype were observed in peripheral T cell lymphomas: one out of six cases showed TcR gene rearrangement, one case retained its germline configuration, a further case masked B cell lymphoma and the remainder were polyclonal. Genotypic analysis was helpful in the analysis of a tumour of mixed T cell and myeloid phenotype which was shown to be germline for TcR and immunoglobulin genes, consistent with a myeloid origin. Two histiocytic tumours were found to have clonal rearrangement of TcR genes. Nine out of 11 B cell tumours showed immunoglobulin gene rearrangement. It is concluded that genetic analyses are useful in the analysis of T cell, histiocytic, and B cell tumours in which an immunoglobulin phenotype cannot be defined.

Phenotypic and genotypic heterogeneity of peripheral T-cell lymphoma.

A series of 21 phenotypically characterised T-cell lymphomas histologically defined as lymphocytic, lymphoblastic, immunoblastic, AILD type, pleomorphic, T-zone and Lennert's T-cell lymphoma, were investigated for T-cell receptor (TcR) and immunoglobulin (Ig) gene rearrangements. Phenotypic analyses of frozen sections and cell suspensions were heterogeneous and in many cases no single T-cell marker recognised all of the malignant cells. Data derived by staining with antibodies reactive with antigens in paraffin embedded tissue were consistent with T NHL in all cases except lymphoblastic lymphoma. TcR gene rearrangements were observed in lymphocytic, lymphoblastic and immunoblastic lymphoma, however, in the remaining 14 phenotypically and histologically defined peripheral T-cell lymphomas, 2 showed rearrangement of TcR gamma and beta genes consistent with T NHL and 2 showed Ig JH rearrangements only, suggestive of either reactive T-cell populations masking cryptic disease or presence of tumour populations with aberrant gene rearrangement and expression of T lineage antigens. No Ig or TcR gene rearrangements were found in the remaining 10 cases, in which morphologically identifiable tumour cells comprised 10-90% of the cell population. In 3/6 cases tested some CD3 positive cells failed to stain with WT31 or beta F1, monoclonal antibodies that recognise determinants on combined TcR gamma beta or TcR beta chains respectively. Whether these cases represent tumours arising from an undetermined cell of origin or polyclonal expansions of T-cells remains to be determined. Our results confirm the phenotypic heterogeneity of histologically defined peripheral T-cell lymphoma and indicate that in these particular histological subtypes gene rearrangement analysis can also yield heterogeneous results which may be unhelpful in determining cell lineage and clonality.