Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.

BACKGROUND: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. MATERIALS AND METHODS: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. RESULTS: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. CONCLUSION: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

Risk factors of rejection after penetrating keratoplasty: a retrospective monocentric study.

PURPOSE: To assess risk factors of rejection after penetrating keratoplasty (PKP). METHODS: This retrospective monocentric study assessed risk factors for rejection in patients who underwent PKP at Montpellier University Hospital between June 2005 and September 2018. Graft and donor data were obtained from our tissue bank in Montpellier. Clinical data of recipients were recorded from medical files. Survival was estimated by the Kaplan-Meir method. Potential risk factors of rejection were assessed by multivariate Cox proportional hazards analysis, estimating hazard ratios (HR) and 95% confidence intervals (CI). RESULTS: Among the 316 consecutive patients (59% male, mean SD] age 52 [17]), 360 eyes underwent PKP. Indications for PKP were bullous keratopathy (27%), infectious keratitis (20%), and keratoconus (15%). The median follow-up was 44 months (IQR 22-73). The overall graft survival and irreversible rejection rate at 5 years were 70% and 29%, respectively. Factors associated with risk of rejection were prior indication for graft rejection (SHR [CI 95%] = 7.8 [2.6-23.1]), trauma (SHR [CI 95%] = 3.6 [1.1-11.7]), and infectious keratitis (SHR [CI 95%] = 2.7 [1.2-11.1]), history of corneal neovascularization (SHR [CI 95%] = 2.1 [1.2-3.8]), hypertonia (SHR [CI 95%] = 2.8 [1.8-4.3]), and mixed sex matching (SHR [CI 95%] = 2.0 [1.01-4.0]). CONCLUSION: The significant risk factors of graft rejection after PKP found in this study agree with those from major international cohorts: prior indication for graft rejection, history of neovascularization and high intraocular pressure. Sex matching donor-recipient is a most recent parameter in the literature confirmed by the present analysis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04791696.

The triglycerides and glucose (TyG) index: A new marker associated with nonalcoholic steatohepatitis (NASH) in obese patients.

AIM: Diagnosis of nonalcoholic steatohepatitis (NASH) relies on liver biopsy. Noninvasive tools would be useful to target patients to refer for a biopsy. We aimed to determine the diagnostic value of the triglycerides and glucose (TyG) index, an insulin-resistance indicator, to predict NASH. METHODS: Our study included grade II-III obese patients aged 18-65 years undergoing bariatric surgery and included in the COMET (COllection of MEtabolic Tissues) biobank (NCT02861781). Liver biopsies performed during bariatric surgery were collected from the biobank along with blood derivatives. Biopsies were analysed according to the steatosis, activity and fibrosis (SAF) scoring system to diagnose NASH, nonalcoholic fatty liver disease (NAFLD), and fibrosis. Logistic regression models were performed to identify factors predicting NASH, NAFLD, and fibrosis. RESULTS: Of 238 analysed subjects (mean age 43±12 years, 33.6% men), 29% had type 2 diabetes. Steatosis was present in 67.2%, while NASH and advanced fibrosis (stage F3) were diagnosed in 18.1% and 2.9% respectively. TyG index was independently associated with NASH (odds ratio (OR): 4.7 [95% confidence interval: 2.3;9.5] P < 0.0001), NAFLD (OR: 2.0 [1.1;3.7] P = 0.03) and stages 2-3 fibrosis (OR: 4.0 [1.5;10.8] P = 0.007). NASH was also predicted by gamma-glutamyl transferase (GGT) with an area under the ROC curve: 0.79 [0.71;0.87 P = 0.04] for GGT and TyG index combined. CONCLUSION: In our cohort of severely obese patients, TyG index, when associated with GGT level, exhibited high diagnostic performance to predict NASH. Although validation in larger populations is needed, this result may be of considerable clinical value to predict need for liver biopsy.

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

Microbial contamination and tissue procurement location: A conventional operating room is not mandatory. An observational study.

BACKGROUND: Standard operating rooms (SOR) are assumed to be the best place to prevent microbial contamination when performing tissue procurement. However, mobilizing an operating room is time and cost consuming if no organ retrieval is performed. In such case, non-operating dedicated rooms (NODR) are usually recommended by European guidelines for tissue harvesting. Performing the tissue retrieval in the Intensive care unit (ICU) when possible might be considered as it allows a faster and simpler procedure. OBJECTIVE: Our primary objective was to study the relationship between the risk of microbial contamination and the location (ICU, SOR or NODR) of the tissue retrieval in heart-beating and non-heart-beating deceased donors. MATERIALS AND METHOD: We retrospectively reviewed all deceased donors' files of the local tissue banks of Montpellier and Marseille from January 2007 to December 2014. The primary endpoint was the microbial contamination of the grafts. We built a multivariate regression model and used a GEE (generalized estimating equations) allowing us to take into account the clustered structure of our data. RESULTS: 2535 cases were analyzed involving 1027 donors. The retrieval took place for 1189 in a SOR, for 996 in a hospital mortuary (NODR) and for 350 in an ICU. 285 (11%) microbial contaminations were revealed. The multivariate analysis found that the location in a hospital mortuary was associated with a lower risk of contamination (OR 0.43, 95% CI [0.2-0.91], p = 0.03). A procurement performed in the ICU was not associated with a significant increased risk (OR 0.62, 95% CI [0.26-1.48], p = 0.4). CONCLUSION: According to our results, performing tissue procurement in dedicated non-sterile rooms could decrease the rate of allograft tissue contamination. This study also suggests that in daily clinical practice, transferring patients from ICU to SOR for tissue procurement could be avoided as it does not lead to less microbial contamination.

Assessing the Impact of Mechanical Damage on Full-Thickness Porcine and Human Skin Using an In Vitro Approach.

For most xenobiotics, the rates of percutaneous absorption are limited by diffusion through the horny layer of skin. However, percutaneous absorption of chemicals may seriously increase when the skin is damaged. The aim of this work was to develop an in vitro representative model of mechanically damaged skins. The epidermal barrier was examined following exposure to a razor, a rotating brush, and a microneedle system in comparison to tape-stripping which acted as a reference. Excised full-thickness skins were mounted on a diffusion chamber in order to evaluate the effect of injuries and to mimic physiological conditions. The transepidermal water loss (TEWL) was greatly increased when the barrier function was compromised. Measurements were made for all the damaged biopsies and observed histologically by microscopy. On human and porcine skins, the tape-stripping application (0 to 40 times) showed a proportional increase in TEWL which highlights the destruction of the stratum corneum. Similar results were obtained for all cosmetic instruments. This is reflected in our study by the nonsignificant difference of the mean TEWL scores between 30 strips and mechanical damage. For a specific appreciation, damaged skins were then selected to qualitatively evaluate the absorption of a chlorogenic acid solution using fluorescence microscopy.

Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

Experimental intracameral injection of vancomycin microparticles in rabbits.

PURPOSE: To evaluate the in vivo toxicity and efficacy of previously developed poly-(lactide-co-glycolide)-vancomycin-based microparticles (V-MPLs) for eventual use for endophthalmitis prophylaxis during cataract surgery. METHODS: The intraocular vancomycin concentration profile was evaluated after V-MPL injection into the anterior chamber of rabbit eyes. The toxicology of V-MPLs versus MPLs alone was tested by corneal cellular counting and retinal histology. The prophylactic efficacy of the V-MPLs was evaluated by bacterial counts after introducing contaminated intraocular lenses (IOLs) together with the V-MPLs into one anterior chamber of phakic rabbit eyes or without V-MPLs in control rabbit eyes. RESULTS: Intraocular V-MPLs produced effective vancomycin concentrations over at least 6 hours. Corneal counts revealed no significant increase in dead cells. Retinal toxicity manifested as inflammation 3 hours after injection, reaching its maximum between 12 hours and 24 hours, decreasing by 48 hours, and completely disappearing at 72 hours. Inflammation was similar between V-MPLs and MPLs. Untreated eyes implanted with highly infected IOLs showed severe, reproducible endophthalmitis. No sign of infection was observed with infected IOLs and concomitant V-MPL treatment, supported by bacterial counts showing a significant decrease in colony-forming Staphylococcus epidermidis units in the anterior chamber and on the implant surfaces within 6 hours. CONCLUSIONS: The present study demonstrated the release and toxicologic properties of the authors' newly developed V-MPLs in vivo. In addition, the rabbit model shows that V-MPLs are effective in reducing the risk of experimental endophthalmitis.

Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures.

PURPOSE: To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells. METHODS: Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry. RESULTS: The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent. CONCLUSIONS: These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.

Variations in the characteristics of keratocytes in culture in relation to their location in human cornea.

To reconstruct artificial stroma close to corneal stroma, it is necessary to use keratocytes with high proliferative potential that maintain the keratocyte phenotype as characterised by CD34. To select such cells, we tested the proliferative potential and characterised the keratocytes isolated from 4 different areas of the human cornea: superior perilimbal, inferior perilimbal, superior central and inferior central. Keratocytes isolated from these different areas had significantly different growth rates (p<0.05), as measured by population doublings: superior perilimbal (42.59+/-11.78) > inferior perilimbal (38.23+/-12.67) > superior central (35.69+/-8.07) > inferior central (25.35+/-7.63). Their clonogenic potential evolved in the same order. Moreover, CD34 labelling gave higher levels in the central areas in relation to the perilimbal areas. We found the best location for isolating keratocytes for stromal reconstruction. The superior perilimbal area had the greatest capacity for proliferation, as well as the best clonogenic potential and the average CD34 level (70%) remained high.

Reconstructed corneas: effect of three-dimensional culture, epithelium, and tetracycline hydrochloride on newly synthesized extracellular matrix.

PURPOSE: To evaluate the influence of the 3-dimensional collagen-glycosaminogycan-chitosan (CGC 3D) scaffold, epithelialization, and the addition of tetracycline hydrochloride on the ultrastructural organization, measured by the diameter and spacing of newly synthesized collagen I fibrils. METHODS: Little is known about the role of interactions between epithelial cells and fibroblasts in controlling the extracellular matrix of the cornea. We developed a hemicornea from a CGC 3D matrix cocultured with keratocytes and human epithelial cells. The keratocytes colonized this substrate, proliferated, and synthesized the extracellular matrix, reproducing a living stroma equivalent. RESULTS: Without a 3D scaffold, the collagen fibrils produced had an average diameter that was 42.7 nm and sigma = 16.9 nm. In the CGC 3D scaffold, the fibrils had an average diameter of 33.4 nm, with little dispersion (sigma = 6.7 nm), suggesting a greater regulation. The epithelium permitted a significant reduction in fibril diameter and interfibrillar spacing. Tetracycline hydrochloride had no effect on spacing but did have a significant effect on fibril diameter. We found positive interactions between the epithelium and tetracycline hydrochloride on the regulation of collagen fibrils but not on spacing. The presence of epithelium led to the increased formation of collagens I and V in the subepithelial area of the newly formed matrix. Type VI collagen was localized around the keratocytes throughout the matrix. CONCLUSIONS: Epithelialization and the 3D scaffold had a great influence on the diameter of collagen I fibrils.

Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction.

The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellae composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma.

Effect of human corneal keratocytes and retinal pigment epithelial cells on the mechanical properties of micropatterned collagen films.

Collagen-based micropatterned films were seeded with human corneal keratocyte and epithelial cells to study their mechanical properties as tissue engineering substrates. The patterns were in the form of parallel channels with slanted walls. Influence of cell presence, type and growth on the mechanical properties of the films was investigated. Unseeded films showed gradual strength reduction from an initial value of 0.046 N/mm, possibly due to degradation, down to 0.032+/-0.012 N/mm in 2 weeks. Keratocyte growth was found to significantly improve the mechanical behavior of the films upon 1 week of incubation (0.067+/-0.017 N/mm) and the improvement continued gradually over the next 2 weeks. Films seeded with D407 retinal pigment epithelial cells, on the other hand, experienced a decrease (0.023+/-0.011 N/mm), followed by a slight increase in mechanical properties in the 21-day period. A steady increase in the number of keratocytes along the channels, cytoskeleton alignment and extracellular matrix (ECM) secretion restricted to the channels was observed. Increase in strength observed with keratocytes and, to a lesser extent, with the epithelial cells can be attributed to directional ECM synthesis and the orientation of the cells and their cytoskeleton which contribute to the strength in the direction of the channels. This study showed that cell, especially keratocyte, presence compensates for the degradation of collagen films and improve the overall mechanical properties of the engineered tissue.

Influence of keratocytes and retinal pigment epithelial cells on the mechanical properties of polyester-based tissue engineering micropatterned films.

In this paper the mechanical properties of micropatterned polyester films prepared to serve as tissue engineering scaffolds of cornea were examined. Films were prepared by solvent casting of blends of poly(l-lactide-co-d,l-lactide) and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), on a micropatterned silicon template. They were seeded with keratocytes or retinal pigment epithelial cells and subjected to tensile testing to assess the contribution of cells and the deposited extra-cellular matrix (ECM) to the mechanical properties of the scaffold. In all the tests, the films used were wet and the cells were not fixed. Cell-free scaffolds showed a gradual deterioration in strength upon incubation in the cell culture medium at 37 degrees C; the deterioration rate was highest in the first week and decreased significantly over the second and third weeks. The ultimate strength of the cell-free scaffolds decreased from 0.99 to 0.42N/mm after 21 days of incubation. Cell seeded scaffolds showed a more complicated mechanical strength profile. Their response was found to depend both on the extent of surface coverage and on the cell type. The results were examined after dividing the data into two groups of lower and higher stiffness. For keratocyte seeded scaffolds, the strength of the high stiffness groups continued to increase as the incubation period increased while the lower stiffness groups did not show a distinct change. For the keratocyte seeded scaffolds, tensile strength increased from 0.65N/mm on Day 7 to 0.73N/mm on Day 21. On the other hand, the scaffolds seeded with retinal pigment epithelial cells showed a gradual deterioration over time in both the higher and lower stiffness groups. For epithelial cell seeded scaffolds this was 0.98N/mm on Day 7 and decreased to 0.77N/mm on Day 21 still an improvement over the unseeded scaffolds. This most probably was a result of a lower rate of ECM secretion in comparison to keratocytes and the newly secreted ECM could not compensate for the influence of scaffold degradation on the mechanical properties. It could, therefore, be concluded that cell seeding plays a positive role in strengthening a tissue engineered construct, and cell type has a significant influence on the extent of this improvement.

Major endothelial loss from corneas in organ culture: importance of second endothelial count.

PURPOSE: The aim of this study was to show that major losses can still occur on corneas judged suitable for grafting at the first count. In addition, we studied the frequency of these losses on 1992 corneas over a period of 4 years to evaluate the risk incurred. METHODS: We evaluated the incidence of these major losses and the associated risk factors. An Ishigawa diagram was created with the Cornea Bank team and the ophthalmologists involved in organ retrieval. Endothelial losses caused by bacterial or fungicidal contamination were excluded from the study. For the 29 corneas that suffered major losses, we analyzed the donor files for donor age, clinical file, geographical origins of the corneas, the person who did the retrieval, the length of time the cornea was stored, the data resulting from examining the endothelium at the bank by optical microscope, and the method used for sterilizing the material used. Specific analyses in cases of major loss of endothelial content: anatomopathologic examination of the corneas and search for the herpes simplex virus (HSV; type 1 or 2) by polymerase chain reaction (PCR). We carried out a statistical analysis using a chi(2) test on the 1992 corneas studied to see if the presence of diabetes (type 1 or 2) in the donor led to reduction levels different from those of corneas originating from nondiabetic donors. RESULTS: The incidence was evaluated at between 0.4% and 3% of corneas sampled, and the associated risk factor was between 0.8% and 6% of grafted corneas. The occurrence of major losses was independent of donor age and was independent of the person who did the retrieval. The occurrence of major losses was independent of geographical origin. We tested our media for endotoxin before use and found levels from 0.22 to 3.9 UI/mL. We verified the absence of a chronological relationship between the batches of media used in the bank and the number of major losses observed, showing that the pyrogenicity limit was independent of cytotoxicity limits. Data analysis showed no difference in reduction levels between diabetic and nondiabetic donors (P < 0.05). Results on the detection of HSV-1 by PCR on the storage media were all negative, and these results agree with the anatomopathologic examinations that showed no signs of viral infection. CONCLUSION: Total endothelial losses amounted to 1.4%/yr. Without the double endothelial counts, we would have had 29 primary graft rejections over that period. During storage, this loss has not been linked to a specific cause, but risk factors such as traumatic death, herpes infections, and badly controlled endotoxin levels should be considered when taking preventative actions. For the moment, a second endothelial count before grafting should be carried out, because all these problem grafts conformed to grafting criteria after the first count. The possibility of carrying out this second count is one of the recognized advantages of storage in organ culture.

Development of an optimised culture medium for keratocytes in monolayer.

UNLABELLED: Our objective was to formulate a medium for monolayer culture optimising both keratocyte growth and preservation of the keratocyte phenotype. METHODS: An experimental matrix selected 14 media to test, using 7 components. Selection criteria were growth rates over 5 passages and expression of the CD34 marker. RESULTS: Acetylcholine, insulin and vitamin C had no effect on growth and differentiation. The DMEM + Ham F12 1 : 1 based medium was selected for its initial effect on growth. At concentrations of 5 ng/ml, b-FGF improved the percentage of CD34+ cells without reducing growth rates. New-born calf serum (NCS) had a greater effect on growth than foetal calf serum (FCS). We showed three major interactions: between b-FGF and IGF-1, FCS and IGF-1 and NCS and b-FGF. CONCLUSION: We selected the following medium, which provides optimal growth and preservation of the CD34+ phenotype: DMEM/HAM-F12 + 10% NCS + 5 ng/ml b-FGF + antibiotics.

Reducing contamination when removing and storing corneas: a multidisciplinary, transversal, and environmental approach.

PURPOSE: The combination of a shortage of cornea grafts in France and a national average contamination rate of 9% to 10%, has led us to search for the origins of this contamination. The objective of our study was to reduce the number of unusable grafts resulting from contamination of corneas in organ culture. METHODS: An external audit was carried out by an independent pharmacist on the removal conditions and treatment procedures for corneas. An environmental study was carried out, consisting of microbiological sampling of the corneas of donors who just died (<24 hours) as well as water and air samples in the premises used for removal. The Cornea Bank's procedures were submitted to a microbiological risk analysis using the "failure mode effects and criticity analysis" (FMECA) method. RESULTS: The critical contamination periods were found to be before removal, during mortuary washing and during decontamination of the conjunctival cul-de-sac at the removal stage. The corrective measures taken have reduced contamination rates by half in 1 year. CONCLUSION: Highlighting the sources of contamination has led to the implementation of effective targeted and low-cost measures that have allowed us to reduce significantly the number of cornea graft losses as a result of bacterial and fungal contamination.