Future Perspectives in Small-Diameter Vascular Graft Engineering

The increased demands of small-diameter vascular grafts (SDVGs) globally has forced the scientific society to explore alternative strategies utilizing the tissue engineering approaches. Cardiovascular disease (CVD) comprises one of the most lethal groups of non-communicable disorders worldwide. It has been estimated that in Europe, the healthcare cost for the administration of CVD is more than 169 billion €. Common manifestations involve the narrowing or occlusion of blood vessels. The replacement of damaged vessels with autologous grafts represents one of the applied therapeutic approaches in CVD. However, significant drawbacks are accompanying the above procedure; therefore, the exploration of alternative vessel sources must be performed. Engineered SDVGs can be produced through the utilization of non-degradable/degradable and naturally derived materials. Decellularized vessels represent also an alternative valuable source for the development of SDVGs. In this review, a great number of SDVG engineering approaches will be highlighted. Importantly, the state-of-the-art methodologies, which are currently employed, will be comprehensively presented. A discussion summarizing the key marks and the future perspectives of SDVG engineering will be included in this review. Taking into consideration the increased number of patients with CVD, SDVG engineering may assist significantly in cardiovascular reconstructive surgery and, therefore, the overall improvement of patients’ life.

Stem cells: Haemobiology and clinical data summarising: A critical review

Stem cells (SC) are the unique and "key-cells" in the human body "working" as a source of producing a large number (proliferation) of mature (differentiation) cells inside different tissues ("cytopoiesis") - while at the same time maintaining the ability to "reproduce" themselves (self-renewal). These events are balanced by interactive signals from the extracellular matrix, as well as microenvironment provided by stromal cells. On the other hand, SC plasticity (so-called "inter-systemic plasticity") is the ability of the most "primitive" (immature) adult SCs to switch to novel identities. The phrase SC plasticity also involves phenotypic potential of these cells, broader than spectrum of phenotypes of differentiated cells in their original tissues. Recent increasing clinical use of cell-mediated therapeutic approaches has resulted in enlarged needs for both, higher quantity of SCs and improved operating procedures during extracorporeal manipulations. The aim of harvesting procedures is to obtain the best SC yield and viability. The goal of optimised cryopreservation is to minimise cellular thermal damages during freeze/thaw process (cryoinjury). Despite the fact that different SC collection, purification and cryopreservation protocols are already in routine use - a lot of problems related to the optimal SC extracorporeal manipulations are still unresolved. The objective of this paper is to provide an integral review of early haemobiological and cryobiological research in the unlimited "SC-field" with emphasis on their entities, recent cell-concepts, extracorporeal manipulative and "graft-engineering" systems. Their therapeutic relevance and efficacy in "conventional" SC transplants or regenerative medicine will be briefly summarised. Finally, in this paper original results will not be pointed out - related to neither SC transplants nor regenerative medicine - but a light will be shed on some of them.

Robust Cell Selections and Depletions across Various Hematopoietic Cell Fractions on the Clinimacs Plus Instrument

Introduction: Cell enrichment and/or depletion by selection is critical for graft engineering in cellular and gene therapies. The semi-automated CliniMACS Plus Instrument (Miltenyi Biotec) is used in clinical cell processing to enrich or deplete a variety of hematopoietic cells including T, B, monocytes, NK cells, and/or CD34+ hematopoietic stem/progenitor cells (HSPCs). Antibody-conjugated paramagnetic beads attach to the desired cells which are separated from the rest of the apheresis or bone marrow product in a closed-system magnetic separation column. Either the selected cells retained in the separation column (positive selection) or those in the "flow-through" fraction (negative selection) may be used for downstream processing. There is sparse data comparing cell selection efficiency and purity across the various selection methods on the CliniMACS Plus Instrument. Further, the effect of donor demographics and other upstream processing events on final viable cell recovery (VCR)/ cell purity (CP) are unknown. This study systematically compared these parameters across various selection methods. Methods: From 2008 to 2018, data were obtained from 45 clinical processing protocols involving 991 consecutive adult and pediatric donor/patient apheresis products or bone marrow harvests, which underwent cell selections on the Miltenyi CliniMACS Plus device. Cell selections included 1.CD34+ (n=669), 2.CD4+ (n=179), 3.CD3-/56+ (n=79), 4.CD3+/19- (n=6), 5.CD4+/8+ (n=21), and 6.CD25- (n=37) cells. Based on protocol design or logistical needs, apheresis or bone marrow collections were processed/selected on the same day or held overnight for next day processing. Products were also either washed to remove platelets (PW) or loaded onto the selection device directly. Data on donor age, BMI, donor type (autologous vs. allogeneic), sex, race, pre-apheresis peripheral blood cell counts and concentrations, as well as apheresis bag blood counts were evaluated. Outcome variables included post-selection VCR and CP. Correlations and multivariate regression analyses were performed for the largest selection type, i.e CD34+ cell selections. Results: Summary data (means ± 1SD) on method-specific enrichments and depletions are shown in the Table. Median post-selection VCR and CP varied by selection method. VCR was the highest for the CD34+ cell selections (Fig. a). CP was the highest and most consistent for CD4+ selections (Fig. b). Platelet and red cell depletions also varied by selection method and averaged 3.9 ± 1.63 and 2.06 ± 0.74 logs, respectively. Selection procedures used for cell depletion resulted in near complete removal of the undesired cell fractions (Table). In multivariate regression analyses, products processed on the same day (compared to those held overnight) (Fig. c), those that underwent a PW (Fig. d), and had higher pre-apheresis peripheral blood CD34+ absolute cell counts and concentrations (Fig. e) were associated significantly with higher CD34+ CP (adjusted r2=0.2; p<0.0001). Higher pre-apheresis CD34+ concentrations (Fig. f) were associated with a lower CD34+ VCR (15%, CI: 5.6-25.3). Conversely, PW (Fig. g) correlated with higher CD34+ VCR (3.5%; CI: 0.8-6.28). Trends across the study period demonstrated a significant improvement in CD34+ VCR from 2008 to 2017 (Fig. h). Conclusions: This is the largest study, to our knowledge, to systematically summarize and analyze Miltenyi CliniMACS Plus selections for clinical graft engineering. Cell selections on the Miltenyi CliniMACS Plus resulted in robust enrichment with near complete depletion of the undesired cell fractions. CD34+ CP was higher in patients with higher CD34+ cells counts following mobilization. Same day processing, as well as PW further improved CD34+ CP. PW also improved CD34+ VCR. This was likely due to a reduction in non-specific platelet binding to the paramagnetic beads. Higher pre-selection CD34+ cell concentrations in the product marginally worsened VCR. This was possibly due to bead saturation in each selection method. Improved CD34+ VCR over time was likely due to increased platelet washes in the latter years. Further studies are ongoing to assess impact of cell selection variations on downstream manufacturing steps (cell transduction, expansion) and clinical (cell engraftment, GVHD incidence) outcomes. Disclosures Larochelle: Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.