Hermann Eichler

Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood.

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full‐term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi‐lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.

Cord blood as a source of autologous RBCs for transfusion to preterm infants.

BACKGROUND: This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the blood supply for preterm infants.

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets

Background and Objectives  The INTERCEPT Blood System for Platelets utilizes amotosalen–HCl (S‐59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function.

Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.

BACKGROUND Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.

Fully automated processing of buffy-coat–derived pooled platelet concentrates

BACKGROUND:  The OrbiSac device, which was develo‐ped to automate the manufacture of buffy‐coat PLT con‐centrates (BC‐PCs), was evaluated.

Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood.

BACKGROUND:  Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation.

Viability does not necessarily reflect the hematopoietic progenitor cell potency of a cord blood unit: Results of an interlaboratory exercise

BACKGROUND: Clinical transplant outcome with umbilical cord blood (UCB) as source of hematopoietic progenitor cells (HPCs) is, among other factors, determined by the total number of viable nucleated cells and/or CD34+ cells in the unit. Quantitative and qualitative losses by processing and cryopreservation and by thawing and washing before transfusion may occur, however. Another reason for a discrepancy between the number of cells in the unit released by the cord blood bank and found in the transplant center may be technical differences in cell counting methods between the two sites.

The Mannheim Cord Blood Project: Experience in Collection and Processing of the First 880 Banked Unrelated Cord Blood Transplants

Background: Placental blood (= cord blood, CB) as a source of hematopoietic progenitor cells is an alternative to bone marrow or to peripheral blood stem cells for hematopoietic reconstitution after a myeloablative conditioning regime. With allogeneic CB transplants it is possible to cure patients with life-threatening malignant and nonmalignant diseases. The Mannheim Cord Blood Bank started the processing of unrelated cord blood transplants for routine clinical application in 1996. Materials and Methods: With a signed informed consent of the mothers more than 880 placental blood samples have now been collected by puncturing the umbilical cord of the placenta in situ. CB is only collected when there is evidence of neither maternal infection nor deformity of the child. After collection the CB is stored at room temperature until further processing. Units with a volume of >60 ml or >5 × 108 total nucleated cells (NC) are processed as potential transplants. All specimens are evaluated for NC count, CD34-positive cells, erythroblasts and NC differentiation, HLA class I/II typing, bacterial contamination, AB0/Rh blood groups, and clonogenic capacity of hematopoietic progenitor cells. The maternal blood is screened for infectious diseases. To reduce the volume of the transplants, we centrifuged the blood and isolated the buffy coat by top/bottom separation. Finally, the transplant is frozen with 5% final concentration of dimethyl sulfoxide (DMSO) and stored in the vapor phase of liquid nitrogen. Results: We collected 65 ± 23 ml CB containing 6.6 ± 3.9 × 108 NC (mean ± SD). 50% of all collected units had to be discarded for various reasons. The median recovery of NC, mononucleated cells (MNC) and CD34-positive cells achieved with the buffy coat preparation was 92.6%, 97.1% and 85.4%, respectively. Defining a threshold dose of 3.0 × 107 NC/kg body weight (BW) for transplantation, 23% of the transplants were sufficient for the treatment of patients over 30 kg BW. Six months after delivery we asked all mothers for a further blood sample and for information about the development of the child. 49.1% of them responded within 8 weeks. Conclusion: We showed that in a very close collaboration with the obstetrician it is possible to establish a routine collection and processing procedure for cord blood transplants. Buffy coat separation by centrifugation is an effective method to reduce the volume of CB units. We found that a closed system technique is easy to perform and shows good recovery rates of nucleated cells.

Storage Characteristics of Split Double-Dose Platelet Concentrates Derived from Apheresis and Treated with Amotosalen Hydrochloride and UVA Light for Pathogen Inactivation

Background: By inhibiting nucleic acids, photochemical treatment (PCT) using amotosalen hydrochloride (S-59) in combination with UVA light provides a new method for the inactivation of pathogens and leukocytes, potential contaminants of platelet concentrates (PCs). Material and Methods: We evaluated the effect of PCT on function and viability of PCs derived from apheresis over a storage period of 5 days. Double-dose PCs containing 6.5–7.0 × 1011 platelets (n = 8) were collected in 600 ml of approximately 35% autologous plasma and 65% platelet additive solution. Each collection was split into two equal-sized portions for paired analysis. One day after collection, test PCs were mixed with 15 ml of 3 mmol/l S-59 and illuminated with UVA light for 3 min, followed by a 6- to 8-hour incubation in a compound adsorption device (CAD) for the reduction of residual S-59 and unbound photoproducts. In vitro platelet function measurements included platelet count, pH, lactate dehydrogenase (LDH), β-thromboglobulin (β-TG), hypotonic shock response (HSR), extent of shape change (ESC) and the expression of p-selectin. Platelet morphology and ultrastructure were analyzed by light and electron microscopy (EM). Results: We found no statistically significant differences on day 5 of the storage between the control and test platelets for pH, platelet count, p-selectin, ESC, HSR, morphology, and the percentage of lysed platelets analyzed by EM. The level of β-TG was reduced in the test units by the CAD step. Significant differences (p < 0.05) were observed for LDH and HSR immediately after PCT, indicating a slight platelet activation. Until the end of the storage period, LDH increased further in control and test platelets, but the quantitative difference between both products did not vary upon further storage. The effect for HSR was transient as no differences between treated and untreated PCs were detected at the end of the storage period. Conclusion: These in vitro results indicate that PCT with amotosalen and UVA light is applicable for split double-dose platelets derived from apheresis as it leads only to a transient activation of platelets immediately after the procedure, without affecting the function and structure of PCs over the 5-day storage period.

Introduction of a validation concept for a PCR-based Mycoplasma detection assay

BACKGROUND Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.