Cryopreservation and thawing of cells Yokoyama, Wayne M; Thompson, Maria L; Ehrhardt, Rolf O
Current protocols in immunology,
November 2012, Letnik:
Appendix 3
Journal Article
Successful cryopreservation of cells requires not only that the cells be handled in a proper fashion for harvesting with equipment in place to ensure consistency, reproducibility, and sterility, but ...also that a correct choice and amount of cryoprotective agent is added. In general, a controlled freezing rate of 1°C/min is necessary to retain optimal viability of the recovered cells. There are many variations of cell freezing methods in use, including costly electronically regulated control rate freezers, unstandardized, passive isopropyl alcohol freezing containers, and crude rudimentary devices constructed from Styrofoam boxes or paper insulation. However, for the freezing and recovery of cell lines, primary cells, and stem cell cultures, the protocol described in this unit is simple, reproducible, and successful. Not only does it eliminate the need for isopropanol, as well as the costs and hazards associated with its use and disposal, but it provides a uniform method with improved cell viability and recovery.
Abstract Despite considerable regulatory and clinical hurdles, the development and use of cell-based therapies are gaining momentum. As more of these therapies move toward commercial approval and ...larger-scale distribution, associated manufacturing and processing technologies are being advanced. Modern technologies directed at downstream processing seek to distribute such therapies from the manufacturing site to the patient more efficiently and reliably. Novel small-scale downstream solutions boost the transformation of cell therapies from abstraction to reality.
Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen ...storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel.
There is a tacit assumption that any object in direct contact with crushed ice must rapidly equilibrate near 0 degree C. Yet target temperature and well-to-well temperature consistency are difficult ...to obtain by placing microtiter plates directly on crushed ice. In addition, arranging plates directly on ice presents a number of physical drawbacks including plate instability, variation in the distribution of the plate contact, potential for ice contamination of the well contents, degradation of support integrity with time, and difficulty in leveling the plate.
The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in ...local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled-rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled-rate freezers. The protocol also eliminates the requirement for isopropanol, avoiding the hazards, on-going cost, and inconsistencies associated with its use and disposal. It provides a clinically relevant, inexpensive, reliable, and user-friendly method that successfully prepares cells for long-term cold storage and ensures maximum levels of cell viability post thaw.
Background: Nuclear factor-κB (NF-κB) is a key transcriptional regulator of inflammatory bowel disease (IBD). Aim: To investigate the therapeutic potential of a locally administered “non-viral” ...nuclear factor-κB decoy (NFκBD) in multiple experimental models of IBD. Methods: A fully phosphorothioated decoy oligonucleotide with improved stability that specifically binds NF-κB and blocks inflammatory mediators regulated by this transcription factor without the help of viral envelope-assisted delivery was developed. The therapeutic effects of NFκBD were studied in the trinitrobenzene sulphonic acid, oxazolone and dextran sodium sulphate induced colitis models. Results: Intracolonic administration of NFκBD results in the delivery of NFκBD to inflammatory cells and a reduction of NF-κB heterodimers. In the T helper cell 1-driven trinitrobenzene sulphonic acid-induced colitis model, mice receiving NFκBD treatment exhibit a dose-dependent reduction in disease severity and a more rapid recovery to normal body weight, similar to a clinically relevant dose of budesonide. Clinical efficacy was corroborated by considerable reductions in colitis pathology and tissue levels of several pro-inflammatory markers, including tumour necrosis factor α, interleukin 6, interleukin 1β and monocyte chemotactic protein 1. NFκBD also mitigates disease activity in the T helper cell 2-like oxazolone colitis and epithelial injury-related acute dextran sodium sulphate colitis models. Interestingly, restoration of tissue homeostasis is observed in NFκBD-treated animals with the rapid re-emergence of functional goblet cells and a return to normal patterns of cell proliferation in the mucosal epithelium and smooth muscle cell layers. Conclusions: These data support the potential use of “naked” NFκBD as a cross-functional therapeutic in IBD, and show for the first time that it can facilitate the restoration of colon homeostasis and function.
Wegener's granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung, and kidney. Since T cells make up a significant proportion of cells infiltrating ...granulomatous lesions in WG, we investigated the proliferative response and cytokine profile of T cells from these patients. PBMCs were isolated from 12 patients with active WG, 7 patients with inactive disease, and 12 healthy normal donors. PBMCs from clinically active WG patients exhibited increased proliferation following stimulation with either PMA/ionomycin or anti-CD2 and anti-CD28, when compared with normal donors. In addition, these PBMCs exhibited increased secretion of IFN-gamma, but not of IL-4, IL-5, or IL-10. Furthermore, TNF-alpha production from PBMCs and CD4+ T cells isolated from patients with WG was elevated, when compared with healthy donors. In further studies, we investigated the ability of WG patients' monocytes to produce IL-12 and showed that both inactive and active patients produced increased amounts of IL-12. Finally, the in vitro IFN-gamma production by WG PBMC is inhibited in a dose-dependent manner by exogenous IL-10. These data suggest that T cells from WG patients overproduce IFN-gamma and TNF-alpha, probably due to dysregulated IL-12 secretion, and that IL-10 may therefore have therapeutic implications for this disease.
Atopic dermatitis (AD) is a common chronic skin inflammatory disease. Long-term use of topical corticosteroids in skin inflammation poses risks of systemic and local side effects. The NF-κB ...transcription factor family plays a central role in the progression and maintenance of AD. This study explores the possibility of using topical NF-κB Decoy as a novel therapeutic alternative for targeting Th1/Th2-driven skin inflammation in experimental AD. A high-affinity, topical NF-κB Decoy developed for human efficacy demonstrates: (i) efficient NF-κB Decoy penetration in pig skin, (ii) NF-κB Decoy nuclear localization in keratinocytes and key immune cells, and (iii) potent “steroid-like” efficacy in a chronic dust-mite antigen skin inflammation treatment model. NF-κB Decoy exerts its anti-inflammatory action through the effective inhibition of essential regulators of inflammation and by induction of apoptosis of key immune cells. Unlike betamethasone valerate (BMV), long-term NF-κB Decoy treatment does not induce skin atrophy. Moreover, topical NF-κB Decoy, in contrast to BMV, restores compromised stratum corneum integrity and barrier function. Steroid withdrawal causes rapid rebound of inflammation, while the NF-κB Decoy therapeutic benefit was maintained for weeks. Thus, topical NF-κB Decoy provides a novel mechanism of reducing chronic skin inflammation with improved skin homeostasis and minimal side effects.
Despite recent successful treatment of murine autoimmune disease with anti-IL-12 mAb, it has not yet been addressed whether anti-IL-12 mAb can also be effective in late stages of disease and whether ...it can provide lasting protection against recurrence, especially during continued presence of autoantigen. We used a newly developed psoriasis model in scid/scid mice, which allows easy tracking of pathogenic T cells, to show that when anti-IL-12 mAb is given for 2 wk (1 mg/wk) in the late stage of severe disease, inflammation is greatly reduced, as measured by ear thickness and histology (scores, 1.1 +/- 0.1 vs 2.0 +/- 0.4). Moreover, prolonged treatment (4 wk) of chronic psoriatic mice with high doses of mAb (1 mg/wk; prolonged active anti-inflammatory treatment (PAAIT)) results in the almost complete resolution of lesions (scores, 0.3 +/- 0.1 vs 2.7 +/- 0.2). Surprisingly, however, despite these significant treatment results, the psoriasis-like lesions return soon after the anti-IL-12 mAb treatment is discontinued. This rapid relapse of disease may be attributed to large populations of activated CD4(+) T cells present in the lymph nodes of PAAIT animals still expressing an effector/memory phenotype (CD45RB(low), L-selectin(low)). Upon stimulation in vitro such PAAIT lymph node cells secrete high amounts of IFN-gamma (129 ng/ml); when transferred into naive scid/scid animals they are able to rapidly induce disease without costimulation. Our data indicates an alternative IL-12-independent pathway for pathogenic Th-1-like cells in vivo during the chronic phase of disease that allows these cells to persist and maintain their pathogenicity in the draining lymph tissue of the autoimmune site.
The demonstrated role of E- and P-selectin ligands in the recruitment of Th1 cells raises the question of tissue specificity determination by pathogenic T cells. We took advantage of the fact that ...chronic Th1-mediated inflammation in the scid/scid CD4+CD45RBhigh T cell transfer model can occur at multiple tissue sites, resembling inflammatory bowel disease in the colon and psoriasis in the skin. We show that the majority of infiltrating effector T cells from psoriatic skin expresses high levels of functional P-selectin ligand (87 +/- 3%), detected by P-selectin-Ig (PIg), while a significantly smaller subset of T cells from colitic lesions expresses this ligand (24 +/- 2%). Similarly, E-selectin ligand is preferentially expressed on CD4+ T cells infiltrating the skin (24 +/- 2%), but only on very few CD4+ T cells infiltrating the colon (CIT; 1.3 +/- 0.8%). In contrast, CD4+ T cells infiltrating the skin express alpha4beta7 at a significantly lower level than CIT (mean fluorescence intensity, 28 vs 61, respectively), although, interestingly, alphaEbeta7 was expressed at high levels on both populations. Analysis of the disease-inducing potential of PIg+ and PIg- CD4+ CIT cells revealed that both populations not only express similar levels of the gut-homing molecule alpha4beta7 (mean fluorescence intensity, 50 vs 56, respectively), but do not differ in their capacity to express IFN-gamma. Furthermore, CIT depleted of cells expressing functional P-selectin ligand were able to induce colitis upon transfer, suggesting that induction of colitis in this model may be independent of E- and P-selectin. These results indicate that adhesion molecule expression and the homing pattern of inflammatory T cells are regulated by the local environment independently of their inflammatory capacity.
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