Kristina Spaniol

Flow cytometry in cancer stem cell analysis and separation

In recent years, a special type of cancer cell—the cancer stem cell (CSC)—has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell‐specific activity of certain ATP‐binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell‐specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry‐based sorting principles and future flow cytometric applications for CSC analysis are also discussed.

Engineering of a Secretory Active Three-Dimensional Lacrimal Gland Construct on the Basis of Decellularized Lacrimal Gland Tissue.

Lacrimal gland (LG) insufficiency is a main cause for severe dry eye leading to pain, visual impairment, and eventually loss of sight. Engineering of transplantable LG tissue with secretory capacity is a desirable goal. In this study, a three-dimensional decellularized LG (DC-LG) scaffold with preserved LG morphology was generated by treatment with 1% sodium deoxycholate and DNase solution using porcine LG tissue. To address clinical applicability, the primary in vitro culture of secretory active LG cells from a small tissue biopsy of 1.5 mm diameter was introduced and compared with an established isolation method by enzymatic digestion. Cells from both isolation methods depicted an epithelial phenotype, maintained their secretory capacity for up to 30 days, and exhibited progenitor cell capacity as measured by aldehyde dehydrogenase-1 activity, side population assay, and colony-forming units. Cells from passage 0 were reseeded into the DC-LG and secretory active cells migrated into the tissue. The cells resembled an LG-like morphology and the constructs showed secretory activity. These results demonstrate the possibility of engineering a secretory competent, three-dimensional LG construct using LG cells expanded from a small tissue biopsy and DC-LG as a matrix that provides the native structure and physiological niche for these cells.

Comparison of Application Systems for Autologous Serum Eye Drops

Abstract Purpose: Autologous serum eye drops are used for therapy of severe ocular surface disorders by patients with visual and manual impairments. Until recently, they were prepared under sterile conditions from open blood sampling systems. Closed blood donation systems simplify production. This study compares handling and costs of a new day dosage vial (“Meise-vial”) and a single-dose tube system (“Maco-tube”) based on closed production systems with conventional dropper bottles. Methods: Nonimpaired volunteers and patients with visual or manual impairment (n = 10 each group) single-handedly tested the applicators filled with 1.5 ml sterile isotonic saline solution. Participants rated convenience of opening the containers and applying eye drops on a scale from 1.0 (very good) to 6.0 (very bad). Number of retrievable drops was counted. Participants were asked which system they prefer, both with and without knowledge of the price for the systems. Results: The median for convenience of opening (eye drop application) was 2.0 (1.0) for Meise-vials, 5.0 (4.0) for Maco-tubes, and 2.0 (2.0) for dropper bottles (p < 0.001). Median number of drops retrieved from the systems was 30.5 (vials), 2 (tubes), and 30 (bottles). Ranking did not differ between nonimpaired and impaired participants. Assuming equal prices, 16 participants chose Meise-vials, 14 dropper bottles, and no tubes. With knowledge of pricing, preference changed (p = 0.001), 20 participants (67%) opted for dropper bottles and 5 (17%) preferred the other containers. Conclusion: Convenience of opening, applying eye drops, and number of drops retrieved was substantially better for dropper bottles and Meise-vials compared with Maco-tubes. Bottles and vials were equally well received. With regard to price, nonimpaired as well as impaired participants preferred dropper bottles. While closed systems simplify production, patients preferred dropper bottles for daily application of autologous serum eye drops for a number of reasons.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland-Derived Mesenchymal Stem Cells.

PURPOSE The application of lacrimal gland-derived mesenchymal stem cells (LG-MSC) for the regeneration of lacrimal gland tissue could result in a novel therapy for dry-eye syndrome. To optimize the culture conditions, the purpose of this study was to evaluate the influence of low oxygen on phenotype, differentiation potential, proliferative, and regenerative capacity of murine LG-MSC. METHODS Murine LG-MSC were cultured in 21% and 5% oxygen and characterized by flow cytometry, cell sorter assisted proliferation-, and colony forming unit-assays. Reactive oxygen species (ROS) levels as well as lineage differentiation were evaluated. The effect of conditioned medium of LG-MSC from both oxygen conditions (CM MSC 21%, respectively, CM MSC 5%) on lacrimal gland epithelial cells (LG-EC) was examined in wound healing and proliferation assays. RESULTS Cells under both culture conditions revealed differentiation potential and presented a MSC-specific flow cytometric phenotype. In 5% oxygen, cells yielded less ROS, showed a stable morphology, higher colony forming potential, and an increased proliferation capacity. Five percent oxygen significantly increased the number of CD44+ LG-MSC. Furthermore, CM MSC 5% significantly enhanced migration and proliferation in LG-EC. CONCLUSIONS In vitro expansion in low oxygen preserves the proliferation capacity and differentiation potential of LG-MSC and increases the effects of conditioned medium on migration and proliferation in LG-EC. Therefore, expansion in low oxygen seems to be an excellent method, to obtain vital MSC. Also, an increased number of LG-MSC expressing CD44 was observed under low oxygen, which might be a valuable marker to identify a potent MSC subpopulation.

Descemet-membrane endothelial keratoplasty in patients with retinal comorbidity-a prospective cohort study

AIM To investigate indications, surgical challenges, and outcome of Descemet-membrane endothelial keratoplasty (DMEK) in patients with retinal comorbidities (RC). METHODS In a prospective cohort study, 8 eyes of 8 DMEK-patients with known RC were compared to 38 eyes of 38 DMEK-patients without RC. The duration of surgery, the degree of difficulty graded by the surgeon, and the complications through DMEK-surgery were analyzed for each patient. The best-corrected visual acuity (BCVA), the endothelial cell count, the intraocular pressure, and the subjective satisfaction was evaluated after a 6-month follow-up. Data were compared applying the non-parametric Wilcoxon-, Chi-square- and Fisheŕs-exact-test with P≤0. 05 as level of significance. RESULTS RC-patients had dry age-related macular degeneration (n=4) or history of pars-plana vitrectomy (n=4). The main indication for DMEK was pain due to bullous keratopathy for the RC-patients (n=7, 88%) and visual impairment due to Fuchs endothelial keratoplasty for the non-RC-patients (n=33, 87%). The BCVA increased for both groups (P=0.01, P<0.001) and all corneas cleared. For the RC-patients, the subjective satisfaction improved significantly (P=0.02). Oil-filling and missing support of the vitreous body complicated surgery in vitrectomized eyes. CONCLUSION DMEK is a favorable technique to treat endothelial disorders even if patients suffer from a retinal comorbidity. By enhancing the corneal clarity, it enables retinal examination or intraocular surgery and increases the patientś satisfaction. However, in vitrectomized or silicone-oil filled eyes, the duration of surgery and degree of complexity are increased. An experienced surgeon should perform DMEK in these patients. CLINICAL TRIAL REGISTRATION NUMBER DRKS00007566.

Evaluation of Decellularized Porcine Jejunum as a Matrix for Lacrimal Gland Reconstruction In Vitro for Treatment of Dry Eye Syndrome

Purpose Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells. Methods To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each. Results Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts. Conclusions SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.

Clinical diagnostics for the tear film and the ocular surface

OSDI (Ocular Surface Disease Index) und DEQ (Dry Eye Questionnaire). Für klinische Studien empfiehlt sich die Verwendung der etablierten Fragebögen OSDI (Schiffman et al. 2000) oder DEQ (Begley et al. 2002), vor allem aufgrund der guten Quantifizierbarkeit des Schweregrades der Symptome. IDEEL‐Fragebogen (Impact of Dry Eye on Everyday Life questionnaire). Der IDEEL‐Fragebogen scheint allerdings in der Erfassung von Auswirkungen des trockenen Auges auf die Lebensqualität dem OSDI überlegen zu sein (Grubbs et al. 2014).

Decellularised conjunctiva for ocular surface reconstruction

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.