The use of human cell based medical products (hCBMPs) in a patient-specific manner for cell therapy purposes raises specific issues pertaining to quality control testing designs for each product under examination . European Community (EC) Directive 2001/83/EC relating to medicinal products for human use defines a hCBMP as a medicinal product which has properties to treat or prevent disease in human beings. Regulations and guidelines for CBMP production follow those of conventional medicinal products [2–4]. The European Parliament Regulation N. 1394/2007 on advanced therapy medicinal products, amending the 2001/83/EC Directive, completed the regulatory setting on advanced therapies to be used in Member States . The manufacturing process of CBMPs has to comply with the principles and guidelines of good manufacturing practice (GMP) for medicinal products for human use published by the European Commission [6, 7]. GMP ensures that products are consistently produced and controlled to the quality standards required for their intended use, from the collection and manipulation of raw materials to the processing of intermediate products, the quality controls, storage, labelling and packaging, and release. In general, when a CBMP enters the clinical development phase, the same principles as those for other medicinal products apply . There should be a careful design and validation of the entire manufacturing process of CBMPs, including cell harvesting, cell manipulation processes, the maximum number of cell passages, and combinations with other components of the product, filling, packaging, etc. In order to ensure product safety and efficacy, each step of the manufacturing process of active substances and supportive components should all be demonstrated, as should be the control of the final product. The quality and safety of the cell preparations should be ensured by implementing a quality system that guarantees the certification and the traceability of every batch of material and supply utilized for the procedures and the correct utilization and cleaning of instruments and locations necessary for stem cell manipulation. Furthermore, the organization structure, qualification and training status of the personnel, and the appropriate equipment, should also comply with current GMP standards. An important aspect of advanced therapies is the necessity to process CBMPs in an aseptic environment, to avoid terminal sterilization which would lead to damage to, and the ineffectiveness of, the cell product. Each manufacturing operation requires an appropriate environmental cleanliness level in the operational state to minimize the risks of particulate or microbial contamination of the product or materials being handled. The application of the GMPs for aseptic production, besides checking all the aspects related to the process, is aimed at minimizing possible contamination factors (personnel, environment, equipment, manufacturing and storage conditions) to ensure the safety of the final product. The quality and safety of advanced therapy products must be maintained throughout their production and quality control (QC) cycle, thus ensuring their final use in the patient. An extensive characterization of the cell therapy product (CTP) should be established in terms of identity, purity, potency and suitability for their intended use. In this context, the cell count, that will indicate the CTP dose, is a potency test. On these bases we validated the cell count method according to the International Conference on Harmonization (ICH) Q2 Guidelines  and European Pharmacopoeia (EP) , taking into account the tests’ accuracy, precision, repeatability, linearity and range. According to the ICH Q2 : “accuracy expresses the closeness of agreement between the value which is accepted as either a conventional true value or as an accepted reference value and the value found; precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from the multiple sampling of the same homogeneous sample under the prescribed conditions; repeatability (also termed intra-assay precision) expresses the precision under the same operating conditions over a short interval of time; linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration of an analyte in the same sample”. There are various manual or automatic, cell count methods. Among the manual cell count methods, the Bürker chamber is seen as the reference method and is the only one described in EP . Alternative manual cell counting chambers include the following: Malazzes, Thoma, Lemaur, Nageotte, Neubauer, Neubauer impaired, Agasse Lafont, and Fuch-Rosenthal. These devices basically differ in the type of ruling that they also have cover glasses of different sizes for the counting chamber. All these double net rulings are made of glass and are not disposable. There are also disposable cell count devices as Kovas slides and Fast Read 102 ® slides. In a GMP setting the latter devices will allow the count chamber to be used only once and then be trashed so that the disposal of waste of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution) can be avoided. Moreover, although some automatic counters are marketed, for GMP settings, their associated software should comply with 21 CFR Part 11 [11–14]. On these bases, our primary aim was to validate a disposable cell count method in GMP conditions, to be included in the Validation Master Plan (VMP) to be submitted to the Regulators, to obtain accreditation of our Cell Factory to produce CTPs. We decided to use a manual cell count and validate, according to GMP rules, a disposable device, Fast Read 102 ®, already used in P3 laboratory by our group.
We therefore sought to evaluate whether Fast Read 102 ® could substitute the Bürker chamber in terms of accurancy. Once this hypothesis was proved, we tested repeatability, linearity and range only using Fast Read 102 ®. Two cell subpopulations were chosen for the validation procedure: mononuclear cells (MNCs), as a prototype of lymphocytes, and mesenchymal stem cells (MSCs), both cell therapy products (CTP) that we will produce for immunotherapy and regenerative medicine. In these settings, we validated viability by Trypan Blue dye. We isolated MNCs and MSCs from whole peripheral blood (wPB) and whole bone marrow (wBM) respectively. We also validated the wPB and wBM cell counts, using Tuerk dye as a red cell lysing solution, to establish the best range of a sample dilution. Finally, the outcome of this experiment served to test whether, having found the dilution range, it might also be suitable for the MNC and MSC counts.