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Te delay between thawing the cells and their first use in an assay, and the need to plan expansion of the culture and timing of passaging to have cells ready for harvest, place constraints


on assay scheduling. Cells from successive harvests have undergone a different number of cell divisions and have a different history of manipulation. Te maintenance of the continuous culture increases the risk of variability and contamination of the stock.


Primary cells – cells that have undergone no, or few, population doublings since isolation from the source tissue – generally retain characteristics closer to their in vivo properties than do immortalized cell lines. Tis makes their use essential for some bioassays. However, variability in the source tissue and the isolation procedure contribute to variability in the assay results, and availability of the source tissue and length of the isolation procedure can constrain assay scheduling.


‘Ready-to-use’ cells


Te disadvantages of maintaining cell stocks in continuous culture have driven the development of cell preparations that can be stored as a bank of uniform aliquots, ready for immediate use in a bioassay. Tese are variously described as:


• Ready-to-use • Ready-to-assay • Assay-ready • Taw-and-use • Taw-and-go


Initially, frozen ready-to-use cells were derived from the cells used in existing bioassays. Freezing and resuscitation protocols were optimized, and the performance of the ready-to-use cells was compared with those from continuous culture to determine whether they could be used as a replacement. Now, when developing a bioassay, it is commonly the aim from the outset to develop, if possible, a protocol using ready- to-use cells. Te cells may be developed in-house or custom-made by a CRO. A further option is the increasing availability of off-the-shelf ready-to-use cells that offer a wide range of target receptors, intracellular signaling systems and readout platforms.


Development of cell banks


Te development work required to create a bank of ready-to-use cells varies, depending particularly on whether it concerns a completely novel system or a modification of an existing system. As with any cells intended for use in assay systems, to permit the cells to be used in a GxP environment, preparation of the bank should follow relevant current guidelines and recommendations [1].


First, freezing media and conditions, and protocols for recovery of the cells, are investigated for acceptable consistency and performance of the cells. Ten the bank is created, normally two-tiered, with a master cell bank and a working cell bank. As with any cell bank, vials are then selected for testing. Tis is likely to include appropriate sterility tests, post-thaw viability and functional performance, including the bioassay response. Even though the cells are intended for use immediately post-thawing, functional testing may include the growth characteristics of the cells.


A major advantage of utilizing a vial of cells directly from the bank is that there is less opportunity for variability to arise in the cells before their addition to the assay system than there would be in cells undergoing extensive manipulation and being cultured for different lengths of time. Tis reduces assay-to-assay variability and means that more of the quality control and system suitability testing can be performed on sample vials, and ahead of the assay, rather than on each cell harvest.


As consistency of the cells at the point of addition to the assay is a major advantage of ready-to-use cells, robustness of the protocol for thawing and for any preparation steps is crucial.


Ready-to-use primary cells


For potency testing of some biopharmaceuticals, it may not currently be possible to use an immortalized cell line and the bioassay will depend on the use of primary cells to assess a biological activity relevant to the clinical mode of action (MOA).


Human umbilical vein endothelial cells (HUVECs) can be prepared in-house from donor tissue or purchased cryopreserved. Under suitable culture conditions, they can retain many of their in vivo response characteristics, but only for a limited number of passages. Such ‘primary’ or ‘low-passage’ HUVECs offer a number of functional responses that can be used to assess the potency of biologicals with a MOA involving responses to vascular endothelial growth factor (VEGF) [2]. However, donor-to-donor variability and the change of characteristics over a short period of time in culture make these cells difficult to use for bioassays in a QC environment. Gazzano-Santoro et al. [3] describe how


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