Material Characterisation and Stratification of Conjunctival Epithelial Cells on Electrospun Poly(ε-Caprolactone) Fibres Loaded with Decellularised Tissue Matrices

The conjunctiva, an under-researched yet incredibly important tissue, plays key roles in providing protection to the eye and maintaining homeostasis of its ocular surface. Multiple diseases can impair conjunctival function leading to severe consequences that require surgical intervention. Small conjunctival defects can be repaired relatively easily, but larger defects rely on tissue grafts which generally do not provide adequate healing. A tissue engineering approach involving a biomaterial substrate capable of supporting a stratified epithelium with embedded, mucin-secreting goblet cells offers a potential solution. As a first step, this study aimed to induce stratification of human conjunctival epithelial cells cultured on electrospun scaffolds composed from poly(ε-caprolactone) (PCL) and decellularised tissue matrix (small intestinal submucosa (SIS) or urinary bladder matrix (UBM)) and held at the air/liquid interface. Stratification, up to 5 cell layers, occurred more frequently on scaffolds containing PCL + UBM. Incorporation of these decellularised tissue matrices also impacted material properties, with significant changes occurring to their fibre diameter, tensile properties, and chemical composition throughout the scaffold structure compared to PCL alone. These matrix containing scaffolds warrant further long-term investigation as a potential advanced therapy medicinal product for conjunctiva repair and regeneration.

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Differential Sensitivity of Differentiated Epithelial Cells to Respiratory Viruses Reveals Different Viral Strategies of Host Infection

Journal of Virology ◽

10.1128/jvi.01271-08 ◽

2008 ◽

Vol 83 (4) ◽

pp. 1962-1968 ◽

Cited By ~ 49

Author(s):

Katherina Goris ◽

Sabine Uhlenbruck ◽

Christel Schwegmann-Wessels ◽

Wiebke Köhl ◽

Frank Niedorf ◽

...

Keyword(s):

Epithelial Cells ◽

Virus Titer ◽

Airway Epithelial Cells ◽

Differential Sensitivity ◽

Airway Epithelial ◽

Cell Layers ◽

Syncytial Virus ◽

Host Infection ◽

Precision Cut Lung Slices ◽

Air Liquid Interface

ABSTRACT To address the initiation of virus infection in the respiratory tract, we established two culture systems for differentiated bovine airway epithelial cells (BAEC). Filter-grown BAEC differentiated under air-liquid interface (ALI) conditions to generate a pseudo-stratified mucociliary epithelium. Alternatively, precision-cut lung slices (PCLS) from the bovine airways were generated that retained the original composition and distribution of differentiated epithelial cells. With both systems, epithelial cells were readily infected by bovine parainfluenza virus 3 (BPIV3). Ciliated cells were the most prominent cell type affected by BPIV3. Surprisingly, differentiated BAEC were resistant to infection by bovine respiratory syncytial virus (BRSV), when the virus was applied at the same multiplicity of infection that was sufficient for infection by BPIV3. In the case of PCLS, infection by BRSV was observed in cells located in lower cell layers but not in epithelial cells facing the lumen of the airways. The identity of the infected cells could not be determined because of a lack of specific antibodies. Increasing the virus titer 30-fold resulted in infection of the ALI cultures of BAEC, whereas in PCLS the ciliated epithelium was still refractory to infection by BRSV. These results indicate that differentiated BAEC are readily infected by BPIV3 but rather resistant to infection by BRSV. Disease caused by BRSV may require that calves encounter environmental stimuli that render BAEC susceptible to infection.

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Conjunctival epithelial cells resist productive SARS-CoV-2 infection

10.1101/2021.12.20.473523 ◽

2021 ◽

Author(s):

Majlinda Lako ◽

Robert M Jackson ◽

Catherine F Hatton ◽

Jarmila S Spegarova ◽

Maria Georgiou ◽

...

Keyword(s):

Epithelial Cells ◽

Viral Entry ◽

Ocular Surface ◽

Cell Types ◽

Productive Infection ◽

Conjunctival Epithelium ◽

Genome Expression ◽

Open Question ◽

Air Liquid Interface ◽

Conjunctival Epithelial Cells

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-Kβ activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.

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GMP-compliant Manufacturing Process of an Advanced Therapy Medicinal Product Based on Conjunctival Epithelial Cells

10.21203/rs.3.rs-122190/v1 ◽

2020 ◽

Author(s):

Marina Bertolin ◽

Stefano Ferrari ◽

Claudia Breda ◽

Barbara Ferrari ◽

Diego Ponzin ◽

...

Keyword(s):

Stem Cells ◽

Quality Control ◽

Stem Cell ◽

Medicinal Product ◽

Manufacturing Process ◽

Manufacturing Processes ◽

Epithelial Stem Cells ◽

Advanced Therapy Medicinal Product ◽

Advanced Therapy ◽

Conjunctival Epithelial Cells

Abstract Background. Conjunctival epithelial stem cell therapy represents a potential and valuable therapeutic option for people suffering from conjunctival disorders. We recently developed a research protocol for the ex vivo cultivation of conjunctival epithelial cells. However, manufacturing and release of any Advanced Therapy Medicinal Product (ATMP) must be designed and planned according to the Good Manufacturing Practices (GMPs) guidelines. GMPs require the development and validation of properly defined manufacturing processes, analysis methods and process validations. Our previous experience with GMP-cultured corneal epithelial stem cells for clinical application on patients with limbal stem cell deficiency led us to set up a protocol for cultivation of conjunctival cells with standards complying with the requests for clinical studies. The major challenge for cell-based products is to develop manufacturing processes while maintaining the critical quality parameters in terms of safety, identity, purity and potency.Results. The manufacturing process was re-designed in order to include all the quality control assays needed for the release of any ATMP, i.e., sterility, morphology, cell viability, dose, cell identity and impurities, potency, lack of pyrogens, mycoplasma and viral detection. Methods and acceptance values were set for all the assays. Quality control assays to evaluate safety and efficacy were also investigated.Conclusion. Here, we describe the main phases of the manufacturing process of a conjunctival stem cell-based product to use in clinical applications. Such characterization is crucial for the preparation of documents and dossiers needed by the competent authorities to start a phase I clinical study on patients with conjunctival disorders. The procedure necessary to reach the marketing authorization of such a new cell-based product is still long, but, if reliable and validated, we believe that, in the near future, patients with conjunctival disorders might have a new treatment based on transplantation of autologous cultured conjunctival epithelial stem cells.

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The Ultrastructure of Monkey Gingival Epithelium

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006009x ◽

1967 ◽

Vol 25 ◽

pp. 16-17

Author(s):

C.N. Sun

Keyword(s):

Epithelial Cells ◽

Macaca Nemestrina ◽

Stratum Granulosum ◽

Solution Ph ◽

Gingival Epithelial Cells ◽

Stratum Spinosum ◽

Cell Layers ◽

Gingival Epithelium ◽

The Individual ◽

Different Thickness

The present study demonstrates the ultrastructure of the gingival epithelium of the pig tail monkey (Macaca nemestrina). Specimens were taken from lingual and facial gingival surfaces and fixed in Dalton's chrome osmium solution (pH 7.6) for 1 hr, dehydrated, and then embedded in Epon 812.Tonofibrils are variable in number and structure according to the different region or location of the gingival epithelial cells, the main orientation of which is parallel to the long axis of the cells. The cytoplasm of the basal epithelial cells contains a great number of tonofilaments and numerous mitochondria. The basement membrane is 300 to 400 A thick. In the cells of stratum spinosum, the tonofibrils are densely packed and increased in number (fig. 1 and 3). They seem to take on a somewhat concentric arrangement around the nucleus. The filaments may occur scattered as thin fibrils in the cytoplasm or they may be arranged in bundles of different thickness. The filaments have a diameter about 50 A. In the stratum granulosum, the cells gradually become flatted, the tonofibrils are usually thin, and the individual tonofilaments are clearly distinguishable (fig. 2). The mitochondria and endoplasmic reticulum are seldom seen in these superficial cell layers.

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Construction of an intestinal mucosa-like structure by growing non-transformed human intestinal primary epithelial cells (HIPEC) on a porcine small intestinal submucosa (SIS) derived fiber matrix

Gastroenterology ◽

10.1016/s0016-5085(01)81590-0 ◽

2001 ◽

Vol 120 (5) ◽

pp. A320-A320

Author(s):

A JOSEPH ◽

L NOCHOMOVITZ ◽

J GRENDELL ◽

A PANJA

Keyword(s):

Epithelial Cells ◽

Intestinal Mucosa ◽

Small Intestinal Submucosa ◽

Porcine Small Intestinal Submucosa ◽

Small Intestinal ◽

Fiber Matrix ◽

Intestinal Submucosa

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The micronucleus cytome assay – A fast tool for DNA damage screening in human conjunctival epithelial cells

The Ocular Surface ◽

10.1016/j.jtos.2021.02.011 ◽

2021 ◽

Vol 20 ◽

pp. 195-198

Author(s):

Katerina Jirsova ◽

Viera Vesela ◽

Pavlina Skalicka ◽

Eva Ruzickova ◽

Johana Glezgova ◽

...

Keyword(s):

Dna Damage ◽

Epithelial Cells ◽

Cytome Assay ◽

Conjunctival Epithelial Cells

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Effect of Alpha-1 Antitrypsin on CFTR Levels in Primary Human Airway Epithelial Cells Grown at the Air-Liquid-Interface

Molecules ◽

10.3390/molecules26092639 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2639

Author(s):

Frauke Stanke ◽

Sabina Janciauskiene ◽

Stephanie Tamm ◽

Sabine Wrenger ◽

Ellen Luise Raddatz ◽

...

Keyword(s):

Epithelial Cells ◽

Human Lung ◽

Liquid Interface ◽

Lung Epithelial Cells ◽

Western Blots ◽

Mesenchymal Transition ◽

Protein Levels ◽

Lung Epithelial ◽

Cftr Protein ◽

Air Liquid Interface

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.

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Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for Use in Humans

Cells ◽

10.3390/cells10081908 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1908

Author(s):

Anna Labedz-Maslowska ◽

Agnieszka Szkaradek ◽

Tomasz Mierzwinski ◽

Zbigniew Madeja ◽

Ewa Zuba-Surma

Keyword(s):

Adipose Tissue ◽

Medicinal Product ◽

Stromal Cells ◽

Manufacturing Process ◽

Cellular Therapy ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Advanced Therapy Medicinal Product ◽

Differentiation Potential ◽

Advanced Therapy

Adipose tissue (AT) represents a commonly used source of mesenchymal stem/stromal cells (MSCs) whose proregenerative potential has been widely investigated in multiple clinical trials worldwide. However, the standardization of the manufacturing process of MSC-based cell therapy medicinal products in compliance with the requirements of the local authorities is obligatory and will allow us to obtain the necessary permits for product administration according to its intended use. Within the research phase (RD), we optimized the protocols used for the processing and ex vivo expansion of AT-derived MSCs (AT-MSCs) for the development of an Advanced Therapy Medicinal Product (ATMP) for use in humans. Critical process parameters (including, e.g., the concentration of enzyme used for AT digestion, cell culture conditions) were identified and examined to ensure the high quality of the final product containing AT-MSCs. We confirmed the identity of isolated AT-MSCs as MSCs and their trilineage differentiation potential according to the International Society for Cellular Therapy (ISCT) recommendations. Based on the conducted experiments, in-process quality control (QC) parameters and acceptance criteria were defined for the manufacturing of hospital exemption ATMP (HE-ATMP). Finally, we conducted a validation of the manufacturing process in a GMP facility. In the current study, we presented a process approach leading to the optimization of processing and the ex vivo expansion of AT-MSCs for the development of ATMP for use in humans.

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Temporomandibular Joint Osteoarthritis: Regenerative Treatment by a Stem Cell Containing Advanced Therapy Medicinal Product (ATMP)—An In Vivo Animal Trial

International Journal of Molecular Sciences ◽

10.3390/ijms22010443 ◽

2021 ◽

Vol 22 (1) ◽

pp. 443

Author(s):

Robert Köhnke ◽

Marcus Oliver Ahlers ◽

Moritz Alexander Birkelbach ◽

Florian Ewald ◽

Michael Krueger ◽

...

Keyword(s):

Stem Cell ◽

Hyaluronic Acid ◽

Medicinal Product ◽

Temporomandibular Joint ◽

Stromal Cells ◽

Stromal Cell ◽

Advanced Therapy Medicinal Product ◽

Animal Trial ◽

Advanced Therapy ◽

Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four weeks after combined mechanical and biochemical osteoarthritis induction in 28 rabbits, therapy was initiated by a single intra-articular injection, randomized into the following groups: Group 1: AB Serum (ABS); Group 2: Hyaluronic acid (HA); Group 3: Mesenchymal stromal cells (STx.); Group 4: Mesenchymal stromal cells in hyaluronic acid (HA + STx.). After another 4 weeks, the animals were euthanized, followed by histological examination of the removed joints. The histological analysis showed a significant increase in cartilage thickness in the stromal cell treated groups (HA + STx. vs. ABS, p = 0.028; HA + ST.x vs. HA, p = 0.042; STx. vs. ABS, p = 0.036). Scanning electron microscopy detected a similar heterogeneity of mineralization and tissue porosity in the subchondral zone in all groups. The single intra-articular injection of a stem cell containing, GMP-compliant advanced therapy medicinal product for the treatment of iatrogen induced osteoarthritis of the temporomandibular joint shows a chondroregenerative effect.

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