WP 3: Metrology support for quality control of tissue engineering and suspension culture
State of the art in the field
In regenerative medicine, transplantation of tissues, to repair or replace damaged tissue, and suspensions of haematopoietic or blood-forming stem cells, to restore cells, for example those destroyed by high doses of chemotherapy or radiation therapy, are important methods. In each case, characterisation of (sorted) cells, e.g. vitality and function, is essential to avoid difficulties or even fatal outcomes when treating patients. Tissue engineering is based on scaffolds used as extracellular matrix (ECM). One aspect to assure a high quality of the tissue product grown in a bioreactor is the supply of well defined progenitor or stem cells of the respective tissue type. Furthermore, to produce large tissue transplants, matrices incorporating a vascular system are needed. During transplantation, the artificial vascular system of the ECM can be connected to the blood circuit of the patient. To successfully apply such a technique, the surfaces of the vascular system of the ECM must be covered by endothelial or progenitor cells, which can be detected in peripheral blood [1]. For the production of tissue engineered products - including the original cells to colonise the ECM - no unified guidelines for quality assurance exist. It is expected, that within this project results will be obtained supporting the establishment of corresponding regulations.
On the other hand, rules for the transplantation of haematopoietic stem cells are described by national [2,3] and international guidelines [4] and an international inter-laboratory survey between clinical laboratories was performed [5] to establish an accepted method based on measurement techniques commercially available. However, agreeing on a standard procedure does only ensure the comparability of results obtained in different laboratories and does not allow to derive the conventional true values and their uncertainties of measurement. Since incorrect or inaccurate measurements of stem cell concentrations might result in fatal outcomes, the development of a reference procedure to determine biological influencing quantities, to reduce the uncertainties and to control clinical laboratories is of great importance.
Identification of circulating endothelial cells (CEC), progenitor endothelial cells (PEC) and haematopoietic stem cells (HSC) requires antibody staining using fluorescently labelled antibodies. Cells being positive with respect to anti-CD34 are assigned as progenitor or stem cells. Measurements are performed by flow cytometry to obtain the statistical precicion required. To determine the concentration, different approaches following the description of the respective manufacturer of the instrument are used. At present, commercial flow cytometers do not allow the determination of the volume of the sample and calibration material provided by the manufacturer has to be added to the sample. Traceability is not ensured, since the user has to rely on the concentration or total number of artificial particles, i.e. fluorescent beads, delivered by the company. Within the project proposed we will develop techniques to enrich (progenitor) endothelial cells and stem cells from periperal blood samples. In addition, a reference procedure to determine the concentration of fluorescently labelled target cells with high precision will be developed. Inclusion of immunological staining for cell identification is a new approach for the determination of reference values for cell concentrations. In addition, the measurements will address issues concerning different influencing quantities like e.g. agglomeration or adhesion on different biomaterial surfaces, the quantification of biological background and monitoring of cell viability. To validate flow cytometric data, (confocal) fluorescence microscopy as well as polymerase chain reaction (PCR) will be applied to characterise flow cytometrically or magnetically sorted cells.
Movement beyond the current state of art
Commercially available instruments for flow cytometric analyses are not suited for rare cell detection. Because of the low concentration of CEC, PEC and HSC, an important aspect is the quantification of cell loss due to adhesion or agglomeration and the determination of the biological background. Experience in rare cell detection and validation was acquired at the PTB for flow cytometric malaria diagnosis [6,7]. By employing advanced microscopic imaging techniques, functional characterisation of (sorted) cells by quantification of fluorescence will be achieved. Besides microscopy, PCR will be used for the identification of (sorted) target cells. Rare cell detection, enrichment and functional characterisation will be complemented by developing measurement procedures to determine concentrations of the target cells in peripheral blood and in suspensions used as supply for tissue scaffolds. To this end, a reference method based on absolute volume measurements and the determination of coincidence corrected numbers of cells will be established. These features are not included in commercially available flow cytometers. To measure cell concentrations employing routine instrumentation, calibration particles have to be used. Since the calibration materials do not mimic the properties of biological samples, the instruments are adjusted with respect to "normal" blood samples and may yield erroneous results for pathological materials. The capability of the PTB will allow development a reference procedure to overcome these problems. Compared to the method for the determination of reference values for erythrocytes developed at the PTB and described in a national standard [8], the inclusion of biological specific markers is mandatory to identify stem cells and endothelial cells. Reference methods for cell counting involving immunological staining are not established and presently investigated to determine platelet concentrations [9].
Scientific tasks
Identification of the target cells and interfering cells using different staining protocols Measurement of titration and kinetics of (antibody) staining reactions to optimise differentiation of positive and negative cells Development of procedures for the enrichment of rare (endothelial, stem) cells from peripheral blood with high purity combining different techniques, i.e. density gradients, magnetic / fluorescence activated cell sorting (MACS / FACS) Establishing gravimetrical dilution procedure to precisely determine the volume fraction of target cells Determination of sensitivity and specificity using serial dilution of target cells in blood samples Development of methods to characterise vitality and function of cells Modification of microscopic techniques to characterise antibody binding capacities of different cells and integral DNA content
Technical risks
Detection and sorting of rare cells as well as measurements of low concentrations is particularly sensitive against different influencing quantities. To avoid unforeseen problems and to obtain cell suspension of high purity suited to colonise EMC or for transplantation, multiple staining procedures to identify interfering populations will be used. Furthermore, microscopically based methods as well as PCR will be used to validate the respective target cells and to identify interfering cell populations.
Expected outputs
patent(s) covering novel techniques and procedures publication of results in scientific journals instrumentation and procedures for quality control of cells in suspensions
Selected References
[1] D.G. Duda et al., A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood, Nature Protocols 2 (2007), 805.
[2] Richtlinien zur Transplantation peripherer Blutstammzellen, Deutsches Ärzteblatt 94, A -15984, 1997.
[3] Richtlinien zur Transplantation von Stammzellen aus Nabelschnurblut (CB = Cord Blood), Deutsches Ärzteblatt 96, A-1297, 1999.
[4] FDA Regulations of Stem-Cell-Based Therapies, New England Journal of Medicine 355, (2006) 1730-1735.
[5] Levering, W.H.B.M. et al., Flow Cytometric CD34+ Stem Cell Enumeration: Lessons from Nine Years' External Quality Assessment Within the Benelux Countries, Cytomerty Part B (Clinical Cytometry) 72B, 178-188, 2007.
[6] Krämer, B.; Grobusch, M.P.; Suttorp, N.; Neukammer, J.; Rinneberg, H.: Relative Frequency of Malaria Pigment-Carrying Monocytes of Nonimmune and Semi-Immune Patients from Flow Cytometric Depolarized Side Scatter. In: Cytometry 45 (2001), 133-140
[7] Grobusch, M.P., Hänscheid, T.; Krämer, B.; Neukammer, J.; May, J.; Seybold, J.; Kun, J.F.J.; Suttorp, N.: Sensitivity of Hemozoin Detection by Automated Flow Cytometry in Non- and Semi-Immune Malaria Patients. In: Clinical Cytometry 55B (2003) 46-51
[8] DIN 58932-3, Determination of the concentration of blood corpuscles in blood; Determination of the concentration of erythrocytes; Reference method, April 1994.
[9] DIN 58932-5, Determination of the concentration of blood corpuscles in blood - Part 5: Reference method for the determination of the concentration of thrombocytes, to be released 2007.
For more information: Dr. Rainer Macdonald
