Optimization of Optical and Mechanical Properties of Real Architecture for 3-Dimensional Tissue Equivalents: Towards Treatment of Limbal Epithelial Stem Cell Deficiency

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The ‘optimal’ RAFT TE was thin, transparent but still handleable and was produced using 0.6 ml of 3 mg/ml collagen. Degradation rates of the ‘optimal’ RAFT TE and HAM were similar. hLE achieved confluency on ‘optimal’ RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure.

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP

Pengaruh Kualitas Pelayanan Dimediasi Kepuasan Pelanggan dan Kepercayaan Pelanggan terhadap Loyalitas Pelanggan pada PT Air Manado

Pembayaran rekening pelanggan tepat waktu sebagai representasi loyalitas pelanggan berperan penting untuk menunjang keberlangsungan hidup Perusahaan publik air seperti PT Air Manado. Peningkatan efisiensi penagihan rekening PT Air Manado dapat dilakukan dengan berbagai upaya antara lain meningkatkan kualitas pelayanan, kepuasan pelanggan dan kepercayaan pelanggan. Penelitian ini bertujuan untuk mengetahui pengaruh kualitas pelayanan terhadap kepuasan pelanggan, kepercayaan pelanggan dan loyalitas pelanggan , untuk mengetahui pengaruh kualitas pelayanan terhadap loyalitas pelanggan melalui kepuasan pelanggan dan kepercayaan pelanggan PT Air Manado. Populasi penelitian ini adalah para pelanggan PT Air Manado, jumlah sampel 161 responden, tenik probability sampling dan metoda pengumpulan data angket terbuka. Metoda Penelitian menggunakan Path Analysis. Hasil penelitian menunjukkan bahwa ada pengaruh: (1) signifikan Kualitas Pelayanan terhadap Kepuasan Pelanggan (2) tidak signifikan Kepuasan Pelanggan terhadap Loyalitas Pelanggan; (3) tidak signifikan Kualitas Pelayanan terhadap Loyalitas Pelanggan (4) signifikan Kepuasan Pelanggan terhadap Kepercayaan Pelanggan; (5) signifikan Kualitas Pelayanan terhadap Kepercayaan Pelanggan; (6) tidak signifikan Kepercayaan Pelanggan terhadap Loyalitas Pelanggan; (7) signifikan Kualitas Pelayanan terhadap Loyalitas Pelanggan dimediasi Kepuasan Pelanggan; (8) tidak signifikan Kualitas Pelayanan terhadap Loyalitas Pelanggan dimediasi Kepercayaan Pelanggan di PT Air Manado. Kata kunci : kualitas pelayanan, kepuasan pelanggan, kepercayaan pelanggan, loyalitas pelangga

Functional Limbal Epithelial Cells Can Be Successfully Isolated From Organ Culture Rims Following Long-Term Storage

PURPOSE. Because of a shortage of fresh corneal tissue for research, it was of interest to investigate the potential of successfully isolating human limbal epithelial cells (hLECs) from organ culture corneal-scleral (OCCS) rims. METHODS. Superficial segments of corneal limbus were dissected and digested using collagenase (0.5 mg/mL, 16 hours at 378C). Cell suspensions were separated into four different growth conditions: corneal epithelial cell medium (CM); CM þ 3T3-Swiss albino cells; stromal stem cell medium (SM); and SM þ 3T3 cells. Colony number, hLEC count, cell density, and colony forming efficiency (CFE) were quantified to assess different growth conditions. The expression profile associated with basal hLECs was assessed by immunofluorescence, and epithelial integrity was measured using our real architecture for 3D tissue (RAFT) corneal tissue equivalent. RESULTS. Human limbal epithelial cells can be successfully isolated from OCCS rims following 4 weeks in storage with an 80.55% success rate with 36 corneal rims. Stromal stem cell medium þ 3T3s provided optimal growth conditions. Colony number, total cell number, and cell density were significantly higher at day 7 in cultures with SM than in CM. There were no significant differences between SM and CM when assessing CFE and the expression profile associated with basal hLECs. Cells maintained in SM were found to produce a higher quality epithelium than that cultured in CM. CONCLUSIONS. Organ culture corneal-scleral rims can be a valuable source for hLEC. Using a combination of collagenase-based isolation and medium designed for stromal stem cell isolation, a high number of good quality hLECs can be cultured from tissue that would have otherwise been ignored

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein

Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture.

Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required

Tissue Engineering the Cornea: The Evolution of RAFT.

Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro