Adipose Tissue-Derived Stem Cell Yield Depends on Isolation Protocol and Cell Counting Method.

In plastic surgery, lipofilling is a frequent procedure. Unsatisfactory vascularization and impaired cell vitality can lead to unpredictable take rates in the fat graft. The proliferation and neovascularization inducing properties of adipose tissue-derived stem cells may contribute to solve this problem. Therefore, the enrichment of fat grafts with stem cells is studied intensively. However, it is difficult to compare these studies because many factors-often not precisely described-are influencing the results. Our study summarizes some factors which influence the cell yield like harvesting, isolation procedure and quantification. Stem cells were isolated after liposuction. Quantification was done using a cell chamber, colony counting, or flow cytometry with changes to one parameter, only, for each comparison. Quantification of cells isolated after liposuction at the same harvesting site from the same patient can vary greatly depending on the details of the isolation protocol and the method of quantification. Cell yield can be influenced strongly by many factors. Therefore, a comparison of different studies should be handled with care.

Research progress on hepatic machine perfusion.

Nowadays, liver transplantation is the most effective treatment for end-stage liver disease. However, the increasing imbalance between growing demand for liver transplantation and the shortage of donor pool restricts the development of liver transplantation. How to expand the donor pool is a significant problem to be solved clinically. Many doctors have devoted themselves to marginal grafting, which introduces livers with barely passable quality but a high risk of transplant failure into the donor pool. However, existing common methods of preserving marginal grafts lead to both high risk of postoperative complications and high mortality. The application of machine perfusion allows surgeons to make marginal livers meet the standard criteria for transplant, which shows promising prospect in preserving and repairing donor livers and improving ischemia reperfusion injury. This review summarizes the progress of recent researches on hepatic machine perfusion.

Optimization of liver glycogen extraction when considering the fine molecular structure.

Liver glycogen is a branched glucose polymer that functions as a blood-sugar buffer in animals. Previous studies have shown that glycogen's molecular structure affects its properties. This makes it important to develop a technique that extracts and purifies a representative sample of glycogen. Here we aim to optimize the sucrose density gradient centrifugation method for preserving glycogen's molecular structure by varying the density of the sucrose solution. The preservation of glycogen's structure involves: 1) minimizing molecular damage and 2) obtaining a structurally representative sample of glycogen. The addition of a 10-minute boiling step was also tested as a means for denaturing any glycogen degrading enzymes. Lower sucrose concentrations and the introduction of the boiling step were shown to be beneficial in obtaining a more structurally representative sample, with the preservation of smaller glycogen particles and decreased glycogen chain degradation.

Surgeon Preferences for Endothelial Keratoplasty in Canada.

PURPOSE: To quantify practice patterns and assess attitudes and barriers to performing Descemet membrane endothelial keratoplasty (DMEK) in Canada. METHODS: An anonymous online survey was distributed to all corneal surgeons included in the Canadian Ophthalmological Society's database. RESULTS: Of 70 listed surgeons, 41 responses were collected (58.6% response rate). Most respondents were practicing in university hospitals (43.9%) or private practice (43.9%) and were involved in residency teaching (77.5%). Most respondents performed DMEK surgery (78%), and most surgeons prepared their own DMEK grafts (62%). Surgeons who were in practice for more than 25 years were less likely to perform DMEK (75% vs. 13%, P = 0.009) and performed fewer corneal transplantation in the previous year (mean 28 vs. 44, P = 0.022). Those who were not performing DMEK reported access to preprepared tissue (77.8%), access to wet laboratory courses (50%), and assistance or mentorship (50%) as common facilitators to start performing DMEK surgery. CONCLUSIONS: DMEK is the preferred surgery for endothelial disease among Canadian corneal surgeons. Eye banks play a key role in increased adoption by ensuring an adequate supply of tissue and prestripping tissue for surgeons new to DMEK to be confident in performing it. Ensuring adequate supply of donor tissue and supplementary surgeon training can ensure that DMEK surgery is widely available in Canada.

Isolation, Culture, Cryopreservation, and Preparation of Umbilical Cord-Derived Mesenchymal Stem Cells as a Final Cellular Product Under Good Manufacturing Practices-Compliant Conditions.

Mesenchymal stem cells have gained popularity in cell-based therapies due to their regenerative capabilities, immunomodulation properties, and paracrine activity through trophic factors. It is of utmost importance to establish clinical-grade procedures for the preparation of the mesenchymal stem cells for clinical applications. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of mesenchymal stem cells derived from umbilical cord as a final product under good manufacturing practices-compliant conditions.

Isolation, Culture, Cryopreservation, and Preparation of Skin-Derived Fibroblasts as a Final Cellular Product Under Good Manufacturing Practice-Compliant Conditions.

Cell-based therapies have become a popular approach in the field of regenerative medicine. Human fibroblast cells, one of the cell types widely used in clinical applications, have been used for skin regeneration and wound healing procedures. Furthermore, they are utilized for aesthetic purposes since fibroblasts lose their abilities such as collagen synthesis with age. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of fibroblasts derived from adult human skin as a final product under good manufacturing practice-compliant conditions.

Latest Advancements in Autologous Breast Reconstruction.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Understand the available donor sites for autologous breast reconstruction. 2. Describe the advantages and limitations of each donor site. 3. Provide a rational, algorithmic preoperative evaluation and approach for patients seeking autologous breast reconstruction. 4. Develop an effective postoperative monitoring system to minimize complications and maximize salvage of microvascular thromboses. SUMMARY: Breast reconstruction remains at the heart of the field of plastic and reconstructive surgery, and it is continuously evolving. Tremendous advances in breast implant technology and supplemental products, particularly acellular dermal matrices, have revolutionized breast reconstruction in the modern era. However, microvascular free flap breast reconstruction has also witnessed profound advancements with exceptionally high success rates, with the ability to provide the most durable and natural breast reconstruction. Although the pendulum oscillates between prosthesis-based reconstruction and autologous tissue, the present synopsis will focus on autologous free flap breast reconstruction from an historical perspective, recent advancements in microsurgery, and the future of autologous breast reconstruction.

Swiss Fetal Transplantation Program and Non-enzymatically Isolated Primary Progenitor Cell Types for Regenerative Medicine.

Primary progenitor cell types adequately isolated from fetal tissue samples present considerable therapeutic potential for a wide range of applications within allogeneic musculoskeletal regenerative medicine. Progenitor cells are inherently differentiated and extremely stable in standard bioprocessing conditions and can be culture-expanded to establish extensive and robust cryopreserved cell banks. Stringent processing conditions and exhaustive traceability are prerequisites for establishing a cell source admissible for further cGMP biobanking and clinical-grade production lot manufacture. Transplantation programs are ideal platforms for the establishment of primary progenitor cell sources to be used for manufacture of cell therapies or cell-based products. Well-defined and regulated procurement and processing of fetal biopsies after voluntary pregnancy interruptions ensure traceability and safety of progeny materials and therapeutic products derived therefrom. We describe herein the workflows and specifications devised under the Swiss Fetal Progenitor Cell Transplantation Program in order to traceably isolate primary progenitor cell types in vitro and to constitute Parental Cell Banks fit for subsequent industrial-scale cGMP processing. When properly devised, derived, and maintained, such cell sources established after a single organ donation can furnish sufficient progeny materials for years of development in translational musculoskeletal regenerative medicine.

GMP-Compliant Production of Human Placenta-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells are one of the most attractive sources for stem cell research and therapy. Their safety and efficacy have been demonstrated in many clinical trials. Because of their low immunogenicity and immunomodulatory properties, allogenic MSCs have been transplanted in different clinical studies. MSCs could be in different adult- and fetal-derived tissues including pregnancy products. Placenta-derived mesenchymal stem cells (PLMSCs) that can be harvested without using any invasive procedures from a discarding tissue are one of the important types of mesenchymal stem cells for therapeutic applications. Stem cell manufacturing for therapeutic applications should be in compliance with the basic principles of good manufacturing practice (GMP). Herein, the current chapter is to describe GMP-compliant production of human PLMSCs, which are suitable for clinical applications.

Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Endometrial Stem Cells for Multiple Sclerosis.

Multiple sclerosis (MS) is the most common cause of neurological diseases. Although, there are some effective medications with regulatory approval for treating MS, they are only partially effective and cannot promote repairing of tissue damage directly which occurs in the central nervous system. Therefore, there is an essential need to develop novel therapeutic approaches for neuroprotection or repairing damaged tissue in MS. Accordingly, cell-based therapies as a novel therapeutic strategy have opened a new horizon in treatment of MS. Each setting in cell therapy has potential benefits. Human endometrial stem cells as an invaluable source for cell therapy have introduced treatment for MS. In this respect, good manufacturing practice (GMP) has a pivotal role in clinical production of stem cells. This chapter tries to describe the protocol of GMP-grade endometrial stem cells for treatment of MS.

GMP-Compliant Human Schwann Cell Manufacturing for Clinical Application.

Schwann cells as glial cells in the peripheral nervous system can participate in neurons protection and forming myelin. Additionally, they are important for nerve pulse conduction supporting along axons. On the other hand, it was demonstrated that they are promising cells for the treatment of demyelinating disorders and also central nervous system damages. Herein, for therapeutic application, Schwann cells should be manufactured based on good manufacturing practice standards to achieve safe and effective clinical products. In this respect, the current chapter tries to introduce a standard protocol for manufacturing of human GMP-compliant Schwann cells for clinical application.

Expert Consensus Guidelines on Minimally Invasive Donor Hepatectomy for Living Donor Liver Transplantation From Innovation to Implementation: A Joint Initiative From the International Laparoscopic Liver Society (ILLS) and the Asian-Pacific Hepato-Pancreato-Biliary Association (A-PHPBA).

OBJECTIVE: The Expert Consensus Guidelines initiative on MIDH for LDLT was organized with the goal of safe implementation and development of these complex techniques with donor safety as the main priority. BACKGROUND: Following the development of minimally invasive liver surgery, techniques of MIDH were developed with the aim of reducing the short- and long-term consequences of the procedure on liver donors. These techniques, although increasingly performed, lack clinical guidelines. METHODS: A group of 12 international MIDH experts, 1 research coordinator, and 8 junior faculty was assembled. Comprehensive literature search was made and studies classified using the SIGN method. Based on literature review and experts opinions, tentative recommendations were made by experts subgroups and submitted to the whole experts group using on-line Delphi Rounds with the goal of obtaining >90% Consensus. Pre-conference meeting formulated final recommendations that were presented during the plenary conference held in Seoul on September 7, 2019 in front of a Validation Committee composed of LDLT experts not practicing MIDH and an international audience. RESULTS: Eighteen Clinical Questions were addressed resulting in 44 recommendations. All recommendations reached at least a 90% consensus among experts and were afterward endorsed by the validation committee. CONCLUSIONS: The Expert Consensus on MIDH has produced a set of clinical guidelines based on available evidence and clinical expertise. These guidelines are presented for a safe implementation and development of MIDH in LDLT Centers with the goal of optimizing donor safety, donor care, and recipient outcomes.

Necroptosis in Hepatosteatotic Ischaemia-Reperfusion Injury.

While liver transplantation remains the sole treatment option for patients with end-stage liver disease, there are numerous limitations to liver transplantation including the scarcity of donor livers and a rise in livers that are unsuitable to transplant such as those with excess steatosis. Fatty livers are susceptible to ischaemia-reperfusion (IR) injury during transplantation and IR injury results in primary graft non-function, graft failure and mortality. Recent studies have described new cell death pathways which differ from the traditional apoptotic pathway. Necroptosis, a regulated form of cell death, has been associated with hepatic IR injury. Receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL) are thought to be instrumental in the execution of necroptosis. The study of hepatic necroptosis and potential therapeutic approaches to attenuate IR injury will be a key factor in improving our knowledge regarding liver transplantation with fatty donor livers. In this review, we focus on the effect of hepatic steatosis during liver transplantation as well as molecular mechanisms of necroptosis and its involvement during liver IR injury. We also discuss the immune responses triggered during necroptosis and examine the utility of necroptosis inhibitors as potential therapeutic approaches to alleviate IR injury.

Consensus minimum hemoglobin level above which patients with myelodysplastic syndromes can safely forgo transfusions.

The anemia of MDS often results in decreased quality of life, which is invoked to justify red cell transfusions; however, there are sparse data regarding the minimum hemoglobin (Hb) at which it is safe to forgo transfusions for patients with no evidence of end-organ damage. This issue is even more important in the COVID-19 era, where decreases in blood donations have stressed the blood supply. In March 2018, using a modified Delphi method, we convened a panel of 13 expert MDS clinicians for three iterative rounds to discuss a minimum safe Hb for this population. While the panel was unable to reach the pre-set consensus of 75% for a specific Hb threshold, there was 100% consensus that it be no greater than 7.5 g/dL. Our data suggest that, given no end-organ effects of anemia, patients with MDS can safely forgo transfusions with a Hb of 7.5 g/dL or higher.

[Photoreceptor cell transplantation for future treatment of retinitis pigmentosa]. / Nouvelle approche thérapeutique pour les rétinites pigmentaires - La transplantation de photorécepteurs dérivés de cellules souches.

In inherited retinal diseases such retinitis pigmentosa, characterized by progressive loss of light sensitive neurons (photoreceptors), cell therapy is now considered as an attractive strategy. Photoreceptor cell replacement would be valuable for restoring function to retinas in a way that is independent from the cause of the disease. With advances in stem cell biology, considerable strides have been made towards the generation of retinal cells, in particular with the development of 3D culture systems allowing the generation of retinal organoids from pluripotent stem cells. In this review, we present a state-of-the art of preclinical strategies conducted in animal models for photoreceptor replacement from stem cell-derived photoreceptors and we discuss the important obstacles to overcome in the future.

TITLE: Nouvelle approche thérapeutique pour les rétinites pigmentaires - La transplantation de photorécepteurs dérivés de cellules souches. ABSTRACT: Dans les maladies dégénératives de la rétine affectant les photorécepteurs, la transplantation de cellules permettant la restauration de la vision est aujourd'hui envisagée. La dernière décennie a vu des progrès remarquables dans la génération de cellules de rétine à partir de cellules souches pluripotentes humaines avec, en particulier, le développement de systèmes de culture en trois dimensions (3D) permettant la génération d'organoïdes de rétine. Dans cette revue, nous faisons un état des lieux sur les stratégies précliniques menées dans des modèles animaux pour le remplacement des photorécepteurs par des photorécepteurs dérivés de cellules souches et présentons les obstacles importants qui restent à être surmontés.

Minimally invasive donor hepatectomy, systemic review.

Liver transplantation is a life-saving therapy for patients with end-stage liver disease. Living donation is a critical source of organs throughout the world. Reducing donor morbidity and mortality is of utmost importance while maintaining access to liver transplantation for recipients. While laparoscopy was more rapidly utilized in donor nephrectomy, laparoscopy has been slower to develop for living donor hepatectomies due to the concerns about hemostasis, safety of the donor and quality of the graft. Pure minimal invasive approach has become a standard of care for left lateral sectionectomy (LLS) for pediatric recipients. In the past few years, a number of centers with significant laparoscopic and living donor experience have reported fully minimally invasive approach to hemi-hepatectomies. In this manuscript we discuss the experiences, lessons learned and path forward for laparoscopic and minimal invasive surgery(MIS) in donor hepatectomies (DH).

[Strengthening the quality control of coronary artery bypass grafting].

The quality control of coronary artery bypass grafting (CABG) is an important prerequisite to the graft patency and the long-term outcomes. The evaluation of target vessel is the basis, the choice of surgical types is the means, the high-quality acquisition of graft harvesting is the guarantee, and the anastomotic method and quality is the core. As the most commonly used quality control tool, intraoperative transit time flow measurement can effectively detect the coronary graft failure caused by anastomotic stenosis and guide to repair of the graft. However, some studies showed that the positive predictive value is low, and the evidence is insufficient for the relationship with the long-term patency rate of grafts. Intraoperative instantaneous flow measurement combined with high-resolution epicardial ultrasound can improve the quality, safety and effectiveness of CABG, which should be an important recommendation for CABG quality control. Once the shape of the grafts and anastomotic ports is abnormal and the blood flow is not satisfied, it needs to adjust or re-anastomose immediately. The quality control of CABG requires comprehensive judgment and individualized measures to ensure the safety and long-term outcome of patients.

Identification of High-Quality Fat Based on Precision Centrifugation in Lipoaspirates Using Marker Floats.

BACKGROUND: Centrifugation creates "graded densities" of fat with varying cellular and biological compositions that influence graft retention. This study aimed to find an accurate method to identify fat fractions that are suitable for implantation. METHODS: Five marker floats (0.925, 0.930, 0.935, 0.940, and 0.945 g/ml) were added to human lipoaspirates that were then centrifuged at 1200 g for 3 minutes to grade the density of centrifuged lipoaspirates. After centrifugation, four fat fractions divided by floats were collected for fat characteristics analysis and in vivo grafting, with Coleman fat as a control. RESULTS: Fat characteristics varied significantly between the centrifuged fat fractions divided by the 0.935-g/ml marker float. Compared with low-quality fat (<0.935 g/ml), high-quality fat (>0.935 g/ml) contains more stromal vascular fraction, adipose-derived stem cells, and extracellular matrix content. Furthermore, adipocytes were found to be significantly smaller in high-quality fat than in low-quality fat, and high-quality fat persisted at a greater volume compared with low-quality fat in vivo at week 12. CONCLUSIONS: High-quality fat contains more stromal vascular fraction cells, extracellular matrix content, and small adipocytes, leading to the highest implant volume retention, whereas low-quality fat contains more fragile large adipocytes, leading to the least volume retention. Marker floats can be used to grade the density of lipoaspirates, with fat greater than 0.935 g/ml recommended as a suitable alternative for implantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.