Utilisation of a Bioreactor for Culture and Expansion of Epithelial Cells without the use of Trypsin or Enzymes.

OBJECTIVE: To develop a bioreactor for automated culture, maintenance, and collection of normal human keratinocytes using an enzyme-free propagation method. METHODS: The culture of normal human epithelial keratinocytes was compared in two culture methods - a study team-developed automated bioreactor utilising an enzyme-free passage method, and a manual culture method. Cell size, glucose utilisation, and the proliferative capacity of the two cultures were evaluated. RESULTS: An automated bioreactor, not using enzymes for passage, but instead using the novel Epithelial Pop Up Keratinocytes (ePUK)1 culture technique, resulted in an extended culture longevity and proliferative capacity in normal primary human keratinocytes. Daughter cells were collected up to three times per day utilising the bioreactor. The daughter cells produced by the bioreactor were smaller than daughter cells produced by the manual culture method. The proliferative capacity and health of the parent monolayer within both the bioreactor and the manual culture flask was dependent upon sufficient glucose availability. Due to the contact inhibition nature of epithelial keratinocytes, the bioreactor enabled the study of an adherent cell type soon after cytokinesis and before the cell has integrated as part of an adherent matrix. CONCLUSION: The study demonstrates that increasing the number of media changes per day as necessary, based on glucose utilisation, is necessary for prolonged longevity and functional productivity of ePUK cultures.

Comparison of two decellularized dermal equivalents.

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature-derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co-culture, separated by a physical barrier to create a cell-free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.

Fabrication of Large Size Ex Vivo-Produced Oral Mucosal Equivalents for Clinical Application.

The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 µm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 µm) to the thinnest (228.6-330.2 µm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.

Differentiation of precursors into parathyroid-like cells for treatment of hypoparathyroidism.

BACKGROUND: Hypoparathyroidism is the most frequent permanent complication of thyroid surgery. Our hypothesis is that human precursor cells in culture can be differentiated into parathyroid cells and used to reconstitute function. Human embryonic stem cells (hESCs) are a stable model to study differentiation into parathyroid-like cells. In prior work, the BG01-hESC line was stimulated to form parathyroid-like cells. This cell line is no longer available, however, and additional studies were needed to confirm and extend prior observations. METHODS: Increasing concentrations of fetal bovine serum and timed exposure to Activin A were used to differentiate H1-hESC into parathyroid-like cells. The potential benefit of Sonic hedgehog exposure on parathyroid-like cell development also was evaluated by serial alterations of culture conditions. Calcium-sensing receptor (CaSR), GCM2, and PTH expression (RT-PCR) and PTH protein secretion (ELISA) were used as markers of differentiated cells. RESULTS: We successfully modified our prior protocol to generate cells that express CaSR, GCM2, and PTH RNA from undifferentiated H1-hESC. The cells also secreted PTH. CONCLUSION: We replicated parathyroid differentiation using H1-hESC cells. Our data advance the project toward in vitro differentiation of precursor cells isolated from individual patients for autotransplantation.

Differentiation of human embryonic stem cells to a parathyroid-like phenotype.

Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.

X-linked recessive atrophic macular degeneration from RPGR mutation.

We mapped a new X-linked recessive atrophic macular degeneration locus to Xp21.1-p11.4 and show allelic involvement of the gene RPGR, which normally causes severe peripheral retinal degeneration leading to global blindness. Ten affected males whom we examined had primarily macular atrophy causing progressive loss of visual acuity with minimal peripheral visual impairment. One additional male showed extensive macular degeneration plus peripheral loss of retinal pigment epithelium and choriocapillaries. Full-field electroretinograms (ERGs) showed normal cone and rod responses in some affected males despite advanced macular degeneration, emphasizing the dissociation of atrophic macular degeneration from generalized cone degenerations, including X-linked cone dystrophy (COD1). The RPGR gene nonsense mutation G-->T at open reading frame (ORF)15+1164 cosegregated with the disease and may create a donor splice site. Identification of an RPGR mutation in atrophic maculardegeneration expands the phenotypic range associated with this gene and provides a new tool for the dissection of the relationship between clinically different retinal pathologies.

Clinical course and visual function in a family with mutations in the RPE65 gene.

OBJECTIVE: To evaluate the phenotype of affected and carrier members of a family with mutations in RPE65 (a retinal pigment epithelium gene). METHODS: RPE65 mutation screening was performed on DNA from 2 affected brothers, 1 unaffected brother, both parents, and 3 surviving grandparents using cycle sequencing. Ophthalmic examinations included ophthalmoscopic fundus examination; visual function testing; 2-color, static, dark-adapted threshold perimetry; and rod electroretinographic a-wave phototransduction analysis. RESULTS: The 2 affected brothers carried RPE65 mutations in compound heterozygous form: a maternal Y368H (1156T-->C) missense mutation and a paternal IVS1 + 5g-->a splice-site mutation. Severe visual deficits and an absence of rod and cone electroretinographic responses were diagnosed in both affected boys before the age of 5 years. Visual acuities of about 20/100 during grade school declined to hand movements by the teenage years, and only a rudimentary peripheral temporal visual field remained by the ages of 25 and 29 years. Both parents had normal central visual function, as measured by visual acuity, contrast sensitivity, color vision, and Humphrey 10-2 fields. However, the 50-year-old father showed hundreds of tiny whitish hard drusen in both eyes and had abnormal peripheral function on dark-adapted perimetry, with extended field defects of 15 to 20 dB outside 30 degrees eccentricity. His rod photoreceptor sensitivity and amplitude, calculated by fitting the rod a waves by a model of activation of phototransduction, were normal, but the flicker electroretinographic response was delayed. CONCLUSIONS: The RPE65 mutations Y368H and IVS1 + 5g-->a present in compound heterozygous form cause severe visual compromise in childhood and progress to nearly total vision loss by the second to third decades of life. The retinal and functional changes in the father carrying a presumed functional null allele suggest that some RPE65 heterozygous carriers may manifest visual symptoms.