Engineering redox sensors into CHO cells enables near-real-time quantification of intracellular redox in bioprocesses.

The production of biologics that treat complex diseases, such as cancer, autoimmune, and infectious disease, requires careful monitoring and control of cell cultures. While bioprocess optimizations have dramatically improved production yields, a lack of analytical tools has made it challenging to identify accompanying intracellular improvements. Intracellular redox can diminish the growth and productivity of biologics-producing cells and adversely impact product quality profiles yet characterizing redox is challenging due to its complex and highly transient nature. In this study, we integrated a fluorescent thiol-based redox biosensor to monitor intracellular redox in one bisAb- and two monoclonal antibody-producing clonal cell lines in a 14-day fed-batch bioreactor. We characterized biosensor functionality using three fluorescence measurement techniques and determined sensor oxidation correlates with the intracellular ratio of reduced (GSH) and oxidized glutathione (GSSG), an important cellular antioxidant. Our fed-batch bioreactor studies showed that sensor expression minimally affected bioprocess outcomes, including growth, productivity, product quality attributes, or intracellular redox attributes, including mitochondrial reactive oxygen species and total cellular GSH levels in all cell lines tested. Biosensor measurements taken throughout the culture revealed that the intracellular environment in these cell lines became more reduced throughout the culture, with the exception of a high pH condition which became more oxidized. Our results demonstrate the potential of using biosensors to monitor intracellular changes in near-real-time with minimal process effects, thus potentially improving future bioprocess optimizations.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Analysis of HOX gene expression and the effects of HOXA9 overexpression in fibroblasts derived from keloid lesions and normal skin.

Keloids are fibroproliferative lesions resulting from an abnormal wound healing process due to pathological mechanisms that remain incompletely understood. Keloids tend to occur more frequently in anterior versus posterior body regions (e.g., ears, face, upper torso); this has been attributed to higher skin tension in those areas, although this has not yet been conclusively proven. Previous studies reported reduced expression of multiple homeobox (HOX) genes in keloid versus normal fibroblasts, suggesting a role for HOX genes in keloid pathology. However, HOX genes are differentially expressed along the anterior-posterior axis. Hypothetically, differential HOX expression may be due to differences in body sites, as matched donor sites are often unavailable for keloids and normal skin. To better understand the basis for differential HOX gene expression in cells from keloids compared with normal skin, we compared HOXA7, HOXA9, HOXC8 and HOXC11 expression in keloid and normal skin-derived fibroblasts from various body sites. When keloid (N = 20) and normal (N = 12) fibroblast cell strains were evaluated, expression of HOXA7, HOXA9 and HOXC8 was significantly lower in keloid versus normal fibroblasts. However, HOX gene expression was lower in fibroblasts from more anterior versus posterior body sites. When keloid and normal cells from similar body sites were compared, differential HOX expression was not observed. To investigate the phenotypic relevance of HOX expression, HOXA9 was overexpressed in keloid and normal fibroblasts. HOXA9 overexpression did not affect proliferation but significantly reduced fibroblast migration and altered gene expression. The results suggest that differential HOX expression may be due to differences in positional identity between keloid and normal fibroblasts. However, HOX genes can potentially regulate fibroblast phenotype, suggesting that differential HOX gene expression may play a role in keloid development in anterior body sites.

Effects of early combinatorial treatment of autologous split-thickness skin grafts in red duroc pig model using pulsed dye laser and fractional CO2 laser.

BACKGROUND AND OBJECTIVE: The use of pulsed dye laser (PDL) and fractional CO2 (FX CO2 ) laser therapy to treat and/or prevent scarring following burn injury is becoming more widespread with a number of studies reporting reduction in scar erythema and pruritus following treatment with lasers. While the majority of studies report positive outcomes following PDL or FX CO2 therapy, a number of studies have reported no benefit or worsening of the scar following treatment. The objective of this study was to directly compare the efficacy of PDL, FX CO2 , and PDL + FX CO2 laser therapy in reducing scarring post burn injury and autografting in a standardized animal model. MATERIALS AND METHODS: Eight female red Duroc pigs (FRDP) received 4 standardized, 1 in. x 1 in. third degree burns that were excised and autografted. Wound sites were treated with PDL, FX CO2 , or both at 4, 8, and 12 weeks post grafting. Grafts receiving no laser therapy served as controls. Scar appearance, morphology, size, and erythema were assessed and punch biopsies collected at weeks 4, 8, 12, and 16. At week 16, additional tissue was collected for biomechanical analyses and markers for inflammatory cytokines, extracellular matrix (ECM) proteins, re-epithelialization, pigmentation, and angiogenesis were quantified at all time points using qRT-PCR. RESULTS: Treatment with PDL, FX CO2 , or PDL + FX CO2 resulted in significantly less contraction versus skin graft only controls with no statistically significant difference among laser therapy groups. Scars treated with both PDL and FX CO2 were visually more erythematous than other groups with a significant increase in redness between two and three standard deviations above normal skin redness. Scars treated with FX CO2 were visually smoother and contained significantly fewer wrinkles. In addition, hyperpigmentation was significantly reduced in scars treated with FX CO2 . CONCLUSIONS: The use of fractional carbon dioxide or pulsed dye laser therapy within 1 month of autografting significantly reduced scar contraction versus control, though no statistically significant difference was detected between laser modalities or use of both modalities. Overall, FX CO2 therapy appears to be modestly more effective at reducing erythema, and improving scar texture and biomechanics. The current data adds to prior studies supporting the role of laser therapy in the treatment of burn scars and indicates more study is needed to optimize delivery protocols for maximum efficacy. Lasers Surg. Med. 50:78-87, 2018. © 2017 Wiley Periodicals, Inc.

Scar formation following excisional and burn injuries in a red Duroc pig model.

Scar research is challenging because rodents do not naturally form excessive scars, and burn depth, size, and location cannot be controlled in human longitudinal studies. The female, red Duroc pig model has been shown to form robust scars with biological and anatomical similarities to human hypertrophic scars. To more closely mimic the mode of injury, recreate the complex chemical milieu of the burn wound environment and enhance scar development, an animal model of excessive burn-induced scarring was developed and compared with the more commonly used model, which involves excisional wounds created via dermatome. Standardized, full-thickness thermal wounds were created on the dorsum of female, red Duroc pigs. Wounds for the dermatome model were created using two different total dermatome settings: ∼1.5 mm and ≥ 1.9 mm. Results from analysis over 150 days showed that burn wounds healed at much slower rate and contracted more significantly than dermatome wounds of both settings. The burn scars were hairless, had mixed pigmentation, and displayed fourfold and twofold greater excess erythema values, respectively, compared with ∼1.5 mm and ≥ 1.9 mm deep dermatome injuries. Burn scars were less elastic, less pliable, and weaker than scars resulting from excisional injuries. Decorin and versican gene expression levels were elevated in the burn group at day 150 compared with both dermatome groups. In addition, transforming growth factor-beta 1 was significantly up-regulated in the burn group vs. the ∼1.5 mm deep dermatome group at all time points, and expression remained significantly elevated vs. both dermatome groups at day 150. Compared with scars from dermatome wounds, the burn scar model described here demonstrates greater similarity to human hypertrophic scar. Thus, this burn scar model may provide an improved platform for studying the pathophysiology of burn-related hypertrophic scarring, investigating current anti-scar therapies, and development of new strategies with greater clinical benefit.

Inflammatory responses, matrix remodeling, and re-epithelialization after fractional CO2 laser treatment of scars.

BACKGROUND AND OBJECTIVE: Fractional CO2 laser therapy has been used to improve scar pliability and appearance; however, a variety of treatment protocols have been utilized with varied outcomes. Understanding the relationship between laser power and extent of initial tissue ablation and time frame for remodeling could help determine an optimum power and frequency for laser treatment. The characteristics of initial injury caused by fractional CO2 laser treatment, the rates of dermal remodeling and re-epithelialization, and the extent of inflammation as a function of laser stacking were assessed in this study in a porcine scar model. MATERIALS AND METHODS: Full-thickness burn wounds were created on female Red Duroc pigs followed by immediate excision of the eschar and split-thickness autografting. Three months after injury, the resultant scars were treated with a fractional CO2 laser with 70 mJ of energy delivered as either a single pulse or stacked for three consecutive pulses. Immediately prior to laser treatment and at 1, 24, 96, and 168 hours post-laser treatment, transepidermal water loss (TEWL), erythema, and microscopic characteristics of laser injury were measured. In addition, markers for inflammatory cytokines, extracellular matrix proteins, and re-epithelialization were quantified at all time points using qRT-PCR. RESULTS: Both treatments produced erythema in the scar that peaked 24 hours after treatment then decreased to basal levels by 168 hours. TEWL increased after laser treatment and returned to normal levels between 24 and 96 hours later. Stacking of the pulses did not significantly increase the depth of ablated wells or extend the presence of erythema. Interleukin 6 and monocyte chemoattractant protein-1 were found to increase significantly 1 hour after treatment but returned to baseline by 24 hours post laser. In contrast, expression of transforming growth factor ß1 and transforming growth factor ß3 increased slowly after treatment with a more modest increase than interleukin 6 and monocyte chemoattractant protein-1. CONCLUSIONS: In the current study, the properties of the ablative zones were not directly proportional to the total amount of energy applied to the porcine scars with the use of triple stacking, resulting in only minor increases to microthermal zone (MTZ) depth and width versus a single pulse. Re-epithelialization and re-establishment of epidermal barrier function were observed in laser treated scars by 48 hours post therapy. Finally, many of the inflammatory genes up-regulated by the laser ablation returned to baseline within 1 week. As a whole, these results suggest that microthermal zones created by FXCO2 treatment re-epithelialize rapidly with the inflammatory response to the laser induced injury largely resolved within 1 week post treatment. Further study is needed to understand the relationship between laser stacking and MTZ properties in human scars in order to evaluate the clinical applicability of the stacking technique. Lasers Surg. Med. 49:675-685, 2017. © 2017 Wiley Periodicals, Inc.

Intermittent glucocorticoid treatment improves muscle metabolism via the PGC1α/Lipin1 axis in an aging-related sarcopenia model.

Sarcopenia burdens the older population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are lacking. The glucocorticoid prednisone remodels muscle metabolism on the basis of frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone administration rescued muscle quality in aged 24-month-old mice to a level comparable to that seen in young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and its cofactor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1α, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed myocyte-specific Lipin1 as a nonredundant factor coaxing PGC1α upregulation to the stimulation of both oxidative and anabolic effects. Our study unveils an aging-resistant druggable program in myocytes for the coordinated rescue of energy and mass in sarcopenia.

The human genetic variant rs6190 unveils Foxc1 and Arid5a as novel pro-metabolic targets of the glucocorticoid receptor in muscle.

Genetic variations in the glucocorticoid receptor (GR) gene NR3C1 can impact metabolism. The single nucleotide polymorphism (SNP) rs6190 (p.R23K) has been associated in humans with enhanced metabolic health, but the SNP mechanism of action remains completely unknown. We generated a transgenic knock-in mice genocopying this polymorphism to elucidate how the mutant GR impacts metabolism. Compared to non-mutant littermates, mutant mice showed increased muscle insulin sensitivity and strength on regular chow and high-fat diet, blunting the diet-induced adverse effects on weight gain and exercise intolerance. Overlay of RNA-seq and ChIP-seq profiling in skeletal muscle revealed increased transactivation of Foxc1 and Arid5A genes by the mutant GR. Using adeno-associated viruses for in vivo overexpression in muscle, we found that Foxc1 was sufficient to transcriptionally activate the insulin response pathway genes Insr and Irs1. In parallel, Arid5a was sufficient to transcriptionally repress the lipid uptake genes Cd36 and Fabp4, reducing muscle triacylglycerol accumulation. Collectively, our findings identify a muscle-autonomous epigenetic mechanism of action for the rs6190 SNP effect on metabolic homeostasis, while leveraging a human nuclear receptor coding variant to unveil Foxc1 and Arid5a as novel epigenetic regulators of muscle metabolism.

Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology.

Keloids are disfiguring scars that extend beyond the original wound borders and resist treatment. Keloids exhibit excessive extracellular matrix deposition, although the underlying mechanisms remain unclear. To better understand the molecular basis of keloid scarring, here we define the genomic profiles of keloid fibroblasts and keratinocytes. In both cell types, keloid-derived cells exhibit differential expression of genes encompassing a diverse set of functional categories. Strikingly, keloid keratinocytes exhibited decreased expression of a set of transcription factor, cell adhesion, and intermediate filament genes essential for normal epidermal morphology. Conversely, they exhibit elevated expression of genes associated with wound healing, cellular motility, and vascular development. A substantial number of genes involved in epithelial-mesenchymal transition were also up-regulated in keloid keratinocytes, implicating this process in keloid pathology. Furthermore, keloid keratinocytes displayed significantly higher migration rates than normal keratinocytes in vitro and reduced expression of desmosomal proteins in vivo. Previous studies suggested that keratinocytes contribute to keloid scarring by regulating extracellular matrix production in fibroblasts. Our current results show fundamental abnormalities in keloid keratinocytes, suggesting they have a profoundly more direct role in keloid scarring than previously appreciated. Therefore, development of novel therapies should target both fibroblast and keratinocyte populations for increased efficacy.

Testing Joiner's theory of suicide in jail inmates: an exploratory study.

Thomas Joiner has proposed an interpersonal theory of suicide in which thwarted belongingness, perceived burdensomeness, and an acquired capability to commit self-harm predict suicidal behavior. In an exploratory study, a questionnaire was administered to 38 jail inmates to test the applicability of this theory to these inmates. Only thwarted belongingness was associated with past suicidal behavior and the inmates' own prediction of the likelihood of future suicidal behavior.

Laser Micropatterning Promotes Rete Ridge Formation and Enhanced Engineered Skin Strength without Increased Inflammation.

Rete ridges play multiple important roles in native skin tissue function, including enhancing skin strength, but they are largely absent from engineered tissue models and skin substitutes. Laser micropatterning of fibroblast-containing dermal templates prior to seeding of keratinocytes was shown to facilitate rete ridge development in engineered skin (ES) both in vitro and in vivo. However, it is unknown whether rete ridge development results exclusively from the microarchitectural features formed by ablative processing or whether laser treatment causes an inflammatory response that contributes to rete ridge formation. In this study, laser-micropatterned and non-laser- treated ES grafts were developed and assessed during culture and for four weeks post grafting onto full-thickness wounds in immunodeficient mice. Decreases in inflammatory cytokine secretion were initially observed in vitro in laser-treated grafts compared to non-treated controls, although cytokine levels were similar in both groups five days after laser treatment. Post grafting, rete ridge-containing ES showed a significant increase in vascularization at week 2, and in collagen deposition and biomechanics at weeks 2 and 4, compared with controls. No differences in inflammatory cytokine expression after grafting were observed between groups. The results suggest that laser micropatterning of ES to create rete ridges improves the mechanical properties of healed skin grafts without increasing inflammation.

Glucocorticoid intermittence coordinates rescue of energy and mass in aging-related sarcopenia through the myocyte-autonomous PGC1alpha-Lipin1 transactivation.

Sarcopenia burdens the elderly population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are missing. The glucocorticoid prednisone remodels muscle metabolism based on frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone rescued muscle quality in aged 24-month-old mice to levels comparable to young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing PGC1alpha and its co-factor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1alpha, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed the myocyte-specific Lipin1 as non-redundant factor coaxing PGC1alpha upregulation to the stimulation of both oxidative and anabolic capacities. Our study unveils an aging-resistant druggable program in myocytes to coordinately rescue energy and mass in sarcopenia.

Light-phase prednisone promotes glucose oxidation in heart through novel transactivation targets of cardiomyocyte-specific GR and KLF15.

Circadian time of intake determines the cardioprotective outcome of glucocorticoids in normal and infarcted hearts. The cardiomyocyte-specific glucocorticoid receptor (GR) is genetically required to preserve normal heart function in the long-term. The GR co-factor KLF15 is a pleiotropic regulator of cardiac metabolism. However, the cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted epigenetic action remain undefined. Here we report that circadian time of intake determines the activation of a transcriptional and functional glucose oxidation program in heart by the glucocorticoid prednisone with comparable magnitude between sexes. We overlayed transcriptomics, epigenomics and cardiomyocyte-specific inducible ablation of either GR or KLF15. Downstream of a light-phase prednisone stimulation in mice, we found that both factors are non-redundantly required in heart to transactivate the adiponectin receptor expression (Adipor1) and promote insulin-stimulated glucose uptake, as well as transactivate the mitochondrial pyruvate complex expression (Mpc1/2) and promote pyruvate oxidation. We then challenged this time-specific drug effect in obese diabetic db/db mice, where the heart shows insulin resistance and defective glucose oxidation. Opposite to dark-phase dosing, light-phase prednisone rescued glucose oxidation in db/db cardiomyocytes and diastolic function in db/db hearts towards control-like levels with sex-independent magnitude of effect. In summary, our study identifies novel cardiomyocyte-autonomous metabolic targets of the GR-KLF15 concerted program mediating the time-specific cardioprotective effects of glucocorticoids on cardiomyocyte glucose utilization.

QED radiative corrections for accelerator neutrinos.

Neutrino oscillation experiments at accelerator energies aim to establish charge-parity violation in the neutrino sector by measuring the energy-dependent rate of νe appearance and νµ disappearance in a νµ beam. These experiments can precisely measure νµ cross sections at near detectors, but νe cross sections are poorly constrained and require theoretical inputs. In particular, quantum electrodynamics radiative corrections are different for electrons and muons. These corrections are proportional to the small quantum electrodynamics coupling α ≈ 1/137; however, the large separation of scales between the neutrino energy and the proton mass (~GeV), and the electron mass and soft-photon detection thresholds (~MeV) introduces large logarithms in the perturbative expansion. The resulting flavor differences exceed the percent-level experimental precision and depend on nonperturbative hadronic structure. We establish a factorization theorem for exclusive charged-current (anti)neutrino scattering cross sections representing them as a product of two factors. The first factor is flavor universal; it depends on hadronic and nuclear structure and can be constrained by high-statistics νµ data. The second factor is non-universal and contains logarithmic enhancements, but can be calculated exactly in perturbation theory. For charged-current elastic scattering, we demonstrate the cancellation of uncertainties in the predicted ratio of νe and νµ cross sections. We point out the potential impact of non-collinear energetic photons and the distortion of the visible lepton spectra, and provide precise predictions for inclusive observables.

Isolation and feeder-free primary culture of four cell types from a single human skin sample.

Four types of primary cells-dermal fibroblasts, dermal microvascular endothelial cells, epidermal keratinocytes, and epidermal melanocytes-can be isolated simultaneously from a single human skin sample, without the use of xenogeneic murine feeder cells. This protocol describes the procedures for isolation of these cells from adult full-thickness skin obtained from surgical discard tissue. The cells isolated using this protocol contain stem cell populations and are competent to form functional skin tissue in three-dimensional reconstructed skin models. For complete details on the use and execution of this profile, please refer to Supp et al. (2002), Boyce et al. (2015), Boyce et al. (2017a), Boyce et al. (2017b), and Supp et al. (2019).

Myofibroblasts Are Not Characteristic Features of Keloid Lesions.

Keloids are disfiguring, scar-like lesions that are challenging to treat, with low response rates to current interventions and frequent recurrence. It has been widely reported that keloids are characterized by myofibroblasts, specialized contractile fibroblasts that express alpha-smooth muscle actin (α-SMA). However, evidence supporting a role for myofibroblasts in keloid pathology is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. This study was undertaken to determine whether myofibroblasts can be considered characteristic of keloids. Methods: Myofibroblasts in tissue sections from keloids, hypertrophic scars (HTSs), and normal skin were localized by α-SMA immunostaining. Expression of α-SMA mRNA (ACTA2 gene) in normal skin and keloid tissue, and in fibroblasts from normal skin, keloid, and HTSs, was measured using quantitative polymerase chain reaction. Results: Normal skin did not exhibit α-SMA-expressing myofibroblasts, but myofibroblasts were identified in 50% of keloids and 60% of HTSs. No significant differences in ACTA2 expression between keloid and normal skin tissue were observed. Mean ACTA2 expression was higher in HTS (2.54-fold, P = 0.005) and keloid fibroblasts (1.75-fold, P = 0.046) versus normal fibroblasts in vitro. However, α-SMA expression in keloids in vivo was not associated with elevated ACTA2 in keloid fibroblasts in vitro. Conclusions: Despite elevated ACTA2 in cultured keloid fibroblasts, myofibroblast presence is not a consistent feature of keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloids. Further research is required to define the mechanisms driving keloid formation for development of more effective therapies.

Redox as a bioprocess parameter: analytical redox quantification in biological therapeutic production.

Engineered Chinese hamster ovary (CHO) cells are the most widely utilized cell line for protein-based therapeutics production at industrial scales. Process development strategies which improve production capacity and quality are often implemented without an understanding of underlying intracellular changes. Intracellular redox conditions drive reactions in pathways critical to biologics production, including bioenergetic and biosynthetic pathways, necessitating methods to quantify redox-related changes. Advances in methods for analytical redox quantification presented here, including bioreactor probes, redox-targeted proteomics, genetically encoded redox-sensitive fluorescent proteins, and biochemical assays, are creating new opportunities to characterize the effects of redox in biologics production. Implementing these methods will lead to enhanced media formulations, improved bioprocess strategies, and new cell line engineering targets and ultimately develop redox into an optimizable bioprocess parameter to improve the yield and quality of these lifesaving medicines.

Fractional CO2 laser micropatterning of cell-seeded electrospun collagen scaffolds enables rete ridge formation in 3D engineered skin.

Rete ridges are interdigitations of the epidermis and dermis of the skin that play multiple roles in homeostasis, including enhancing adhesion via increased contact area and acting as niches for epidermal stem cells. These structures, however, are generally absent from engineered skin (ES). To develop ES with rete ridges, human fibroblast-seeded dermal templates were treated with a fractional CO2 laser, creating consistently spaced wells at the surface. Constructs with and without laser treatment were seeded with keratinocytes, cultured for 10 days, and grafted onto athymic mice for four weeks. Rete-ridge like structures were observed in the laser-patterned (ridged) samples at the time of grafting and were maintained in vivo. Ridged grafts displayed improved barrier function over non-lasered (flat) grafts at the time of grafting and 4 weeks post-grafting. Presence of ridges in vivo corresponded with increased keratinocyte proliferation, epidermal area, and basement membrane length. These results suggest that this method can be utilized to develop engineered skin grafts with rete ridges, that the ridge pattern is stable for at least 4 weeks post-grafting, and that the presence of these ridges enhances epidermal proliferation and establishment of barrier function. STATEMENT OF SIGNIFICANCE: Rete ridges play a role in epidermal homeostasis, enhance epidermal-dermal adhesion and act as niches for epidermal stem cells. Despite their role in skin function, these structures are not directly engineered into synthetic skin. A new method to rapidly and reproducibly generate rete ridges in engineered skin was developed using fractional CO2 laser ablation. The resulting engineered rete ridges aided in the establishment of epidermal barrier function, basement membrane protein deposition and epidermal regeneration. This new model of engineered skin with rete ridges could be utilized as an in vitro system to study epidermal stem cells, a testbed for pharmaceutical evaluation or translated for clinical use in full-thickness wound repair.

Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds.

Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 (KRT20), a general marker for Merkel cells, in the basal epidermis of ESS after transplantation to mice, suggesting the presence of Merkel cells. Cells expressing KRT20 were not observed in ESS in vitro. However, widely separated KRT20-positive cells were observed in basal epidermis of ESS by 2 weeks after grafting. By 4 weeks, these cells increased in number and expressed keratins 18 and 19, additional Merkel cells markers. Putative Merkel cell numbers increased further between weeks 6 and 14; their densities varied widely and no specific pattern of organization was observed, similar to Merkel cell localization in human skin. KRT20-positive cells co-expressed epidermal markers E-cadherin and keratin 15, suggesting derivation from the epidermal lineage, and neuroendocrine markers synaptophysin and chromogranin A, consistent with their identification as Merkel cells. By 4 weeks after grafting, some Merkel cells in engineered skin were associated with immature afferents expressing neurofilament-medium. By 8 weeks, Merkel cells were complexed with more mature neurons expressing neurofilament-heavy. Positive staining for human leukocyte antigen demonstrated that the Merkel cells in ESS were derived from grafted human cells. The results identify, for the first time, Merkel cell-neurite complexes in engineered skin in vivo. This suggests that fine touch sensation may be restored in ESS after grafting, although this must be confirmed with future functional studies.

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures.

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 106 cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2-10 × 106 cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22-34 × 106 cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3-6 × 106 cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.