Danielle E. Dye

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A₂ decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

The ability to grow C2C12 myoblasts in a completely defined, serum free medium enables researchers to investigate the role of specific factors in myoblast proliferation, migration, fusion, and differentiation without the confounding effects of serum. The use of defined, animal free in vitro culture systems will improve reproducibility between research groups and may also enhance translation of tissue engineering techniques into clinical applications. Here, we describe the use and characterization of a serum free culture system for C2C12 myoblasts using standard tissue culture medium and readily available, defined growth factors and supplements.

Introduction: Increased student numbers and limited cadaver/prosected materials in health courses at Curtin University have required reviewing the way in which we teach our students in laboratory classes. The Anatomage table, an interactive tablet for viewing life-size anatomical images, was introduced into first-year human biology classes in 2013 to replace cadaver materials. Methods: Two student cohorts were surveyed in 2013 and 2014 on their perception of the usefulness of the Anatomage table and other anatomy resources to their learning. On a scale of 0–100, students were asked to rate a range of anatomy learning tools, including the Anatomage table. Results: Respondents (2013, n = 333; 2014, n = 329) rated video/animations most useful for learning (77.8/100), followed by models (63.9/100), plastinates (58.4/100) and the Anatomage table (42.4/100), a pattern consistent across gender, use of digital devices and cohort year. In 2014, respondents rated the Anatomage table more favourably (42.4/100) than in 2013 (36.9/100) (p = 0.022). The Anatomage table was rated most helpful for understanding relative sizes of organs but least helpful for using correct anatomical terminology. Qualitative data showed that in 2013, students were frustrated by screen-freezing problems and low-quality graphics, issues that were mostly addressed by 2014. Across both years, student comments were positive regarding the 3D aspect, seeing organ sizes and relationships, the slice tool for cross-sections and avoiding the need for cadaver specimens. Students also liked the Anatomage structured activities and pre-set images but wanted more time to explore and less than eight students around the table at any one time. Students felt unprepared for using the Anatomage table, and the single touch capacity was limiting when working in groups. Conclusions: These findings indicate that touch-screen technology needs careful curriculum design and training for both students and staff to optimise its usefulness for learning.

The average age of the human population is rising, leading to an increasing burden of age‐related diseases, including increased susceptibility to infection. However, immune function can decrease with age which could impact on processes that require a functional immune system. Aging is also characterized by chronic low‐grade inflammation which could further impact immune cell function. While changes to neutrophils in blood during aging have been described, little is known in aging lymphoid organs. This study used female C57BL/6J mice comparing bone marrow (BM), spleen and lymph nodes from young mice aged 2–3 months (equivalent to 18 human years) with healthy elderly mice aged 22–24 months (equivalent to 60–70 human years). Neutrophil proportions increased in BM and secondary lymphoid organs of elderly mice relative to their younger counterparts and presented an atypical phenotype. Interestingly, neutrophils from elderly spleen and lymph nodes were long lived (with decreased apoptosis via Annexin V staining and increased proportion of BrdU^neg mature cells) with splenic neutrophils also demonstrating a hypersegmented morphology. Furthermore, splenic neutrophils of elderly mice expressed a mixed phenotype with increased expression of activation markers, CD11b and ICAM‐1, increased proinflammatory TNFα, yet increased anti‐inflammatory transforming growth factor‐beta. Elderly splenic architecture was compromised, as the marginal zone (required for clearing infections) was contracted. Moreover, neutrophils from elderly but not young mice accumulated in lymph node and splenic T‐ and B‐cell zones. Overall, the expansion of functionally compromised neutrophils could contribute to increased susceptibility to infection observed in the elderly.

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

Aging is associated with a decline in lymphocyte function however, little is known about dendritic cell (DC) subsets and aging. Aging is also associated with increasing circulating lipid levels and intracellular lipid accumulation modulates DC function. Whether age-associated increases in lipid levels influence DC biology is unknown. Thus, the effects of aging on DC subsets were assessed in vivo using young adult and elderly C57BL/6 J mice.

The role of macrophages and their interactions with T cells during aging is not well understood. We determined if activating elderly-derived macrophages could rescue age-related and tumor-induced T cell dysfunction. Healthy elderly (18–24 months) Balb/c contained significantly more splenic IL-10-secreting M2-macrophages and myeloid-derived suppressor cells than young (6–8 weeks) mice. Exposure to syngeneic mesothelioma or lung carcinoma-conditioned media polarized peritoneal macrophages into suppressive M2-macrophages regardless of age. Tumor-exposed, elderly, but not young-derived, macrophages produced high levels of IL-4 and could not induce T cell IFN-γ production. We attempted to rescue tumor-exposed macrophages with LPS/IFN-γ (M1 stimulus) or IL-2/agonist anti-CD40 antibody. Tumor-exposed, M1-stimulated macrophages retained high CD40 expression, yet TNF-α and IFN-γ production were diminished relative to non-tumor-exposed, M1-stimulated controls. These macrophages induced young and elderly-derived T cell proliferation however, T cells did not secrete IFN-γ. In contrast, tumor-exposed, IL-2/CD40-stimulated macrophages rescued elderly-derived T cell IFN-γ production, suggesting that IL-2/CD40-activated macrophages could rescue T cell immunity in aging hosts.