Research paper
Purified enzymes improve isolation and characterization of the adult thymic epithelium

https://doi.org/10.1016/j.jim.2012.07.023Get rights and content

Abstract

The reproducible isolation and accurate characterization of thymic epithelial cell (TEC) subsets is of critical importance to the ongoing study of thymopoiesis and its functional decline with age. The study of adult TEC, however, is significantly hampered due to the severely low stromal to hematopoietic cell ratio. Non-biased digestion and enrichment protocols are thus essential to ensure optimal cell yield and accurate representation of stromal subsets, as close as possible to their in vivo representation. Current digestion protocols predominantly involve diverse, relatively impure enzymatic variants of crude collagenase and collagenase/dispase (col/disp) preparations, which have variable efficacy and are often suboptimal in their ability to mediate complete digestion of thymus tissue. To address these issues we compared traditional col/disp preparations with the latest panel of Liberase products that contain a blend of highly purified collagenase and neutral protease enzymes. Liberase enzymes revealed a more rapid, complete dissociation of thymus tissue; minimizing loss of viability and increasing recovery of thymic stromal cell (TSC) elements. In particular, the recovery and viability of TEC, notably the rare cortical subsets, were significantly enhanced with Liberase products containing medium to high levels of thermolysin. The improved stromal dissociation led to numerically increased TEC yield and total TEC RNA isolated from pooled digests of adult thymus. Furthermore, the increased recovery of TEC enhanced resolution and quantification of TEC subsets in both adult and aged mice, facilitating flow cytometric analysis on a per thymus basis. We further refined the adult TEC phenotype by correlating surface expression of known TEC markers, with expression of intracellular epithelial lineage markers, Keratin 5 and Keratin 8. The data reveal more extensive expression of K8 than previously recognized and indicates considerable heterogeneity still exists within currently defined adult TEC subsets.

Introduction

The development of T cells from hematopoietic stem cell (HSC) – derived lymphoid precursors, requires fundamental interactions with resident thymus stromal cells (TSC) which govern the processes of T cell differentiation, selection and maturation (Takahama, 2006). Specialized thymic stromal cells dictate self -tolerance induction (Anderson et al., 2007); accordingly defects in the microenvironment predispose for T cell dysregulation and autoimmune-like disease (Fletcher et al., 2011). Furthermore, the thymic stroma is critically implicated in the process of age-related thymic atrophy, and alongside the aging bone-marrow, is an important facet of immunosenescence (Dudakov et al., 2010). Unraveling the role and function of TSC is thus fundamental to our understanding, not only of thymopoiesis, but also for the development of strategies to combat states of immune-dysfunction and disease.

Despite their importance, a major obstacle in the study of TSC is their reproducible isolation from the thymus organ. Contrary to most epithelial-based tissues, the thymus is organized as a unique, three dimensional (3-D) network of adjoining TEC and non-TEC stromal cells to facilitate maximal thymocyte migration throughout defined cortical and medullary compartments (van Ewijk et al., 1999). The stroma is held together by extensive connections within the extracellular matrix (Lannes-Vieira et al., 1991) and thus requires extended enzymatic digestion to release cells into suspension. In particular, the study of adult stroma is further impeded by their rarity within mature thymus tissue (comprising < 1% of total thymic cellularity) (Gray et al., 2006), and their inherently fragile nature — particularly in the cortex where the TEC have long and slender cytoplasmic processes (Hoshino, 1963). A combination of gentle enzymatic digestion steps and enrichment protocols is thus required to effectively isolate TSC with minimal cellular damage and maximal yield to ensure accurate phenotypical and functional analysis.

Parameters such as enzymatic constituents, purity and concentration, as well as the time taken to digest tissue, all impact upon the final cell yield and viability of digested tissues and organs. Traditionally, crude enzymatic preparations of collagenase from Clostridium Histolyticum have been used for the isolation of TSC (Kyewski and Kaplan, 1982, Lahoud et al., 1993, Izon et al., 1994). These preparations are often variable, with significant enzymatic contaminants and impurities, such as endotoxin, that critically impact upon cell isolates (Vargas et al., 1998, Eckhardt et al., 1999). Liberase research grade enzymes are a blend of highly purified collagenase and neutral protease enzymes that are designed to improve the quality and reproducibility of primary tissue cell isolates. In comparison to traditional collagenase preparations, Liberase enzymes contain considerably higher collagenolytic activity and negligible endotoxin content and have reported benefits in the isolation of cells from a number of primary organs and tissues (Linetsky et al., 1997, Dolmans et al., 2006, Brandhorst et al., 2010).

While use of proteases, such as trypsin, allow more complete digestion of thymic tissue, cleavage of important epithelial epitopes hinders their use for the isolation of TEC subsets (Gray et al., 2002). At present, current thymus literature suggests that crude enzymatic preparations of collagenase are still in wide use for the isolation and study of TSC phenotype and function (Bonfanti et al., 2010, Cheng et al., 2010, Corbeaux et al., 2010, Goldberg et al., 2010, Kvell et al., 2010, Osada et al., 2010, Santos et al., 2010, Sun et al., 2010, McLelland et al., 2011, Rode and Boehm, 2012). Here, we directly compare traditional preparations of collagenase with the latest panel of Liberase enzymes for their ability to effectively isolate viable TSC, characterize TEC and subsets thereof, and for total TEC RNA obtained from pooled thymic digests to facilitate downstream molecular analysis.

Section snippets

Animals

Unless otherwise specified, adult (6–8 weeks) and aged (9 months) male C57Bl/6 mice were used in this study. Mice were obtained from Monash University Central Animal Services and maintained at Animal Research Laboratories (Monash University, Clayton, Melbourne, Aust.) according to institutional and ethical guidelines.

Antibodies and immunoconjugates

The following primary biotinylated or fluorochrome-conjugated antibodies were used in this study. Antibodies were purchased from BD Pharmingen™ (U.S.A) unless otherwise stated.

Liberase enzymes improve digestion of adult thymus tissue and increase viable thymic epithelial cell recovery

To assess the efficacy of discreet enzyme products to mediate digestion of murine adult (6–8 weeks) thymus tissue, individual thymus digestions (n = 5) were undertaken using a panel of second-generation Liberase products and compared against digestion using crude collagenase D/col/disp enzymes (refer 2.3.1), hereafter referred to as col/disp digestion. Digestion with Liberase enzymes TM and TH were found to rapidly dissociate adult thymic tissue within 3 incubations steps (36 min), whilst Liberase

Conclusion

Reproducible and efficient isolation of TSC, whilst retaining high cell viability, is critical to ensure accurate phenotypic and functional characterization of TEC subsets. Digestion of adult thymus with Liberase enzymes, notably Liberase containing medium to high levels of thermolysin, greatly enhanced the digestion of adult and aged thymus tissue, significantly improving epithelial yield. The improved digestion protocol described here will provide a more comprehensive and accurate platform

Conflict of interest

The authors have no conflict of interest.

Acknowledgements

The authors would like to thank Flowcore (Monash University, Australia) for their expert cell sorting. This work was supported by grants from the Australian Stem Cell Centre, National Health and Medical Research Council and the Victorian Government–Californian Institute for Regenerative Medicine.

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