A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

Flow cytometric sorting is a vital tool in biological research and clinical diagnostics. Theoretically, a high-speed jet-in-air sorter is a fluorescent-activated cell sorting sorter that ideally processes cells with high purity, yield, and viability. However, high-speed jet-in-air sorting is a complex process due to its inherent requirements for high fluidic stability and electronic and timing precision. Here, we report that an additional manual correction of drop delay leads to improved cell yield. Adding 2% FBS to the loading buffer had no significant effect on the fate of sorted cells in 4 h. However, the addition of a suitable concentration of FBS/BSA in the collecting buffer resulted in a notable increase in cell count and proliferation and a significant decrease in cell apoptosis for cell lines and primary cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. In summary, here we demonstrate techniques that can be easily followed to refine sorted yields of healthy cells.

Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application.

B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27+ B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.

Overview of current adipose-derived stem cell (ADSCs) processing involved in therapeutic advancements: flow chart and regulation updates before and after COVID-19.

Adipose-derived stem cells (ADSCs) have raised big interest in therapeutic applications in regenerative medicine and appear to fulfill the criteria for a successful cell therapy. Their low immunogenicity and their ability to self-renew, to differentiate into different tissue-specific progenitors, to migrate into damaged sites, and to act through autocrine and paracrine pathways have been altogether testified as the main mechanisms whereby cell repair and regeneration occur. The absence of standardization protocols in cell management within laboratories or facilities added to the new technologies improved at patient's bedside and the discrepancies in cell outcomes and engraftment increase the limitations on their widespread use by balancing their real benefit versus the patient safety and security. Also, comparisons across pooled patients are particularly difficult in the fact that multiple medical devices are used and there is absence of harmonized assessment assays despite meeting regulations agencies and efficient GMP protocols. Moreover, the emergence of the COVID-19 breakdown added to the complexity of implementing standardization. Cell- and tissue-based therapies are completely dependent on the biological manifestations and parameters associated to and induced by this virus where the scope is still unknown. The initial flow chart identified for stem cell therapies should be reformulated and updated to overcome patient infection and avoid significant variability, thus enabling more patient safety and therapeutic efficiency. The aim of this work is to highlight the major guidelines and differences in ADSC processing meeting the current good manufacturing practices (cGMP) and the cellular therapy-related policies. Specific insights on standardization of ADSCs proceeding at different check points are also presented as a setup for the cord blood and bone marrow.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Optimizing ovarian tissue quality before cryopreservation: comparing outcomes of three decortication methods on stromal and follicular viability.

OBJECTIVE: To evaluate whether specific ovarian decortication techniques vary in promoting ovarian cortex cryopreservation and transplant outcomes. DESIGN: Experimental design. SETTING: University hospital. ANIMAL(S): Nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice. INTERVENTION(S): Human ovarian biopsy samples allocated to one of the following decortication procedures: scratching with scalpel blade (B), cutting with microsurgical scissors (M), separation with slicer (S), or no-separation (control, C). Parallel, in vivo experiment: decortication techniques combined with slow freezing (SF) and vitrification (VT) before xenograft into immunodeficient mice. MAIN OUTCOME MEASURE(S): Follicular counts, apoptosis, shear stress, Hippo pathway and inflammation. In vivo: recovered grafts analyzed for follicular counts, angiogenesis, proliferation, and fibrosis. RESULT(S): There were no differences in follicular density or number of damaged follicles between the decortication techniques in the in vitro study. Nevertheless, the M samples showed statistically significantly increased stromal damage compared with the controls and S samples, and up-regulation of Hsp60 shear stress gene expression. Decortication by both M and S inhibited the Hippo pathway, promoting gene expression changes. In the 21-day xenograft, total follicular density statistically significantly decreased compared with the nongrafted controls in all groups. Nevertheless, no differences were observed between the decortication techniques. Ovarian stroma vascularization was increased in the vitrified samples, but among the slow-freezing samples, the B samples had the lowest microvessel density. The M decorticated xenografts had increased fibrosis. CONCLUSION(S): Decortication with a slicer causes less damage to ovarian tissue than other commonly used methods although microsurgical scissors seem to preserve slightly increased follicular numbers. Nevertheless, blade decortication seems to be a reliable technique for maintaining acceptable follicular conditions without inducing serious stromal impairment.

A more efficient preparation system for HLA-eliminated platelets.

BACKGROUND AND OBJECTIVES: Although HLA-eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow-fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation. MATERIALS AND METHODS: HLA-eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow-fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals. RESULTS: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody-containing sera, suggested that HLA-ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist-induced aggregability and adherence/aggregability of the collagen-coated plates under certain conditions, were conserved and not significantly different from the original ones. CONCLUSION: We propose a novel preparation system for producing HLA-eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality.

Methodological considerations for the high sensitivity detection of multiple myeloma measurable residual disease.

BACKGROUND: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 105 -106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches. METHODS: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. RESULTS: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10-5 and approached that of 10-6 for detecting rare events. CONCLUSION: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.

High-Parameter Immune Profiling with CyTOF.

Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).

Establishment of normative ranges of the healthy human immune system with comprehensive polychromatic flow cytometry profiling.

Existing normative flow cytometry data have several limitations including small sample sizes, incompletely described study populations, variable flow cytometry methodology, and limited depth for defining lymphocyte subpopulations. To overcome these issues, we defined high-dimensional flow cytometry reference ranges for the healthy human immune system using Human Immunology Project Consortium methodologies after carefully screening 127 subjects deemed healthy through clinical and laboratory testing. We enrolled subjects in the following age cohorts: 18-29 years, 30-39, 40-49, and 50-66 and enrolled cohorts to ensure an even gender distribution and at least 30% non-Caucasians. From peripheral blood mononuclear cells, flow cytometry reference ranges were defined for >50 immune subsets including T-cell (activation, maturation, T follicular helper and regulatory T cell), B-cell, and innate cells. We also developed a web tool for visualization of the dataset and download of raw data. This dataset provides the immunology community with a resource to compare and extract data from rigorously characterized healthy subjects across age groups, gender and race.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Fine-tuning blastocyst selection based on morphology: a multicentre analysis of 2461 single blastocyst transfers.

RESEARCH QUESTION: Which blastocyst morphology parameter is associated with live birth after controlling for female age and endometrial receptivity? DESIGN: Retrospective study including fresh single blastocyst transfers (n = 2461) where the value of serum progesterone on day of human chorionic gonadotrophin trigger (PdHCG) was available. Generalized estimating equation regression models evaluated the independent effects of developmental stage (DevSt), inner cell mass (ICM) and trophectoderm grade on live birth rates while controlling for the confounding effects of female age and PdHCG. RESULTS: DevSt was strongly associated with the probability of live birth (P < 0.0001) independently of female age (odds ratio [OR] 0.89, 95% confidence interval [CI] 0.87-0.91) and PdHCG (OR 0.80, 95% CI 0.74-0.87). For full blastocysts, expanded blastocysts and hatching blastocysts, addition of ICM and trophectoderm grading in the multivariable analysis suggested that besides female age (OR 0.92, 95% CI 0.90-0.94) and PdHCG (OR 0.80, 95% CI 0.73-0.87), only DevSt (P = 0.001) and trophectoderm quality (P = 0.004) were independent predictors of live birth, while the predictive capacity of ICM was no longer significant. The mean probability of live birth was highest for AA blastocysts (35.0%), followed by BA blastocysts (31.2%) and AB blastocysts (27.7%). CONCLUSION: This large study analyses for the first time the independent role of blastocyst morphology in predicting live birth while controlling for female age and PdHCG. Its findings suggest that DevSt and then trophectoderm grade are stronger predictors of live birth over ICM grade when selecting a single blastocyst for transfer.

Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting. METHODS: We collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions. RESULTS: The survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product. CONCLUSIONS: Survey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.

Isolation, culturing and preparation for transplantation of amniotic mesenchymal stem cells Repetitive and reproducible laboratory, technical protocol.

To date, different methods of isolation of amniotic stem cells have been developed. Our previous studies have demonstrated that there are significant differences in viability and efficiency of the isolation and culture process depending on the enzyme and medium used. The aim of this study was to present efficient protocol, which can be used within good manufacturing practise conditions. Amniotic membranes were obtained from ten woman 31-39 years old who signed informed constent. GMP regulations are applicable. The described protocol aims to obtain a clinically significant cell yield (>1*108). The cells may be maintained in the growth phase even for 2 months. The mesenchymal cells constitute about 75-95% of the cells in primary culture. Supervisory authorities require repetitive and reproducible laboratory protocol for stem cells culture. Presented protocol allow achieving clinically significant cell yield (>1*108) in 4-5 weeks. Cells can be transplanted as suspension or cell sheet.

The identification and isolation of CTCs: A biological Rubik's cube.

Liquid biopsy represents an alternative to conventional biopsies for the evaluation of tumors mainly due to its easy sampling. One of the main applications is the enumeration of Circulating Tumor Cells (CTCs) to evaluate tumor progression or response to treatment. The analysis of the functional characteristics of CTCs could give us much more information about their role in order to establish a more personalized treatment for the patients. The major issue that has to be solved is the isolation of the CTC population. Multiple protocols have been developed, however none of them has demonstrated to be the definitive one. In fact, a combination of these techniques has often been performed in order to obtain a purer and viable population of CTCs. In this review we have summarized for the first time the different combinatorial approaches used in the last years to optimize the isolation of CTCs and their limitations.

Evaluation of the effect of storage condition on cell extraction and flow cytometric analysis from intestinal biopsies.

BACKGROUND: Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. METHODS: Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. RESULTS: Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. CONCLUSION: Storage of intestinal tissue biopsies at -20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.

Considerations pertaining to cell collection and administration of industry-manufactured autologous CAR-T cells, in relation to French healthcare organization and regulations.

Access to treatment with CAR-T Cells at European hospitals in general and at French hospitals in particular remains limited, when compared with the situation that prevails in the USA or in certain Asian countries. Multiple reasons explain why European investigators lag behind their US or Chinese colleagues in this clinical research area. Some of these reasons are related to the European and French regulatory landscapes that hamper the design and rapid implementation of organizational solutions needed for safe and efficient administration of CAR-T Cells. We here identify some of these pressing issues and propose some possible paths to move forward.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.