Seeding, culturing and assaying upcyte and vericyte cells in the Mimetix 3D scaffold

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1 Seeding, culturing and assaying upcyte and vericyte cells in the Mimetix 3D scaffold Description This note describes how to seed and culture upcyte Hepatocytes as 3D cultures in the Mimetix electrospun scaffold for subsequent endpoint measurements. The protocol can be adapted to all upcyte and vericyte cells by modifying, for example, cell numbers seeded, pre-coating of the scaffolds, etc. Material required In order to conduct the 3D cell culture experiments described in this application note, the following items are required: upcyte Hepatocytes (cryopreserved) Each vial contains ~6 x 10 6 upcyte Hepatocytes (upcyte; upregulated hepatocytes ) frozen per vial from which at least 50% recovery is expected after thawing. upcyte Hepatocyte Thawing Medium Use this medium to thaw upcyte Hepatocytes. upcyte Hepatocyte High Performance Medium The upcyte Hepatocyte High Performance Medium is designed for the optimal culture and endpoint determination using upcyte Hepatocytes. In order to obtain upcyte Hepatocyte High Performance Medium add the entire contents of Supplement A to the basal medium. In addition the medium needs to be supplemented with L- Glutamine to a final concentration of 2 mm. The shelf life of the fully supplemented medium lasts up to 6 weeks. Once fully supplemented do not freeze the upcyte Hepatocyte High Performance Medium. We recommend adding 100 U/mL penicillin and 100 g/ml streptomycin to the medium for these studies. Mimetix 3D scaffolds (96-well format) The Mimetix scaffold is made of the FDAapproved polymer poly-l-lactide (PLLA) with randomly-orientated fibres. Fibre diameters are highly consistent, leading to reproducible results in cell-based assays well-to-well and batch-to-batch. The thickness of the scaffolds (50 µm) facilitates fluorescent microscopic imaging in situ, while providing the benefits of 3D cell culture to observe 3D cell morphology and behaviour. Two different fibre diameters (2 m and 4 m) are available, corresponding to pore sizes of m and m, respectively. Note: The Mimetix scaffold is delivered gamma irradiated, i.e. sterile. Before adding cells, the Mimetix scaffold needs to be wetted with ethanol in order to allow an aqueous solution, such as cell medium, to access the pores. For best results, 200 µl of 20% ethanol is added to each well of a 96-well plate and allowed to soak into the scaffold membrane. This should wet the scaffolds evenly. The ethanol is then aspirated from the wells, being careful not to touch the membrane. Please visit our website for more information on our products, protocols and to order samples. The Electrospinning Company Ltd, R104 Rutherford Appleton Laboratory, Harwell Oxford, Oxfordshire, OX11 0QX, UK E Controlled info@electrospinning.co.uk Document Property T +44 (0)845 of 388 The 8856 Electrospinning Company V1.0

2 Subsequently, each well is washed twice with upcyte Hepatocyte High Performance Medium. In some cases, it can be beneficial to leave the scaffold soaked in the medium for 30 min. Finally the cell suspension is added (see below). The Mimetix scaffold is available in different formats, i.e. 6-well with hanging inserts, 12-well and 96-well. Storage The Mimetix 3D plates can be stored at room temperature; temperatures above 30 C should be avoided. Stor upcyte Hepatocytes can be stored in liquid or vapour phase nitrogen. They should not be stored at -70 C. Store basal medium, as well as fully supplemented upcyte Hepatocyte High Performance Medium, protected from light at 2 to 8 C. Store Supplement A at -20 C. The expiration date is indicated on the label of the basal medium as well as on the supplement label. Experimental Procedure The following steps will describe how to thaw, seed and culture upcyte Hepatocytes in the Mimetix scaffold. Cell-based assays, such as MTS and evaluation of CYP enzyme activity, are conducted according to the instructions of the kit manufacturers. Note: All assays with fluorescence- and luminescence-based readouts can be conducted exactly in the same way than for standard 2D tissue culture plate. Absorbancebased readouts are impaired by the intrinsic background absorbance of the Mimetix scaffold. Thawing of cryopreserved upcyte Hepatocytes This is a shortened version of the full protocol. If more detail is require, the full version (PFU No. 1) can be downloaded at the link below: wnload-center/pfus.html 1. Pre-warm 50 ml upcyte Hepatocyte Thawing Medium to 37 C. 2. Carefully remove the cryovial from the storage tank and thaw cells in a 37 C water bath until all the ice has completely disappeared. Do not shake. 3. Transfer the thawed cell suspension (1 ml) from the cryovial into 50 ml thawing medium by gently pouring the cells into the medium. 4. Transfer 1 ml of the thawing medium back to the cryovial and then back into the 50 ml tube to completely remove the cells from the cryovial. Repeat twice. 5. Pellet the cells by centrifuging at 90 g for 5 min at RT. Carefully aspirate supernatant, leaving μl medium on top of the cells. 6. Gently loosen the cells by agitating and rotating the tube. Add 1 ml of pre-warmed culture medium per million cells and carefully resuspend. Do not shake/vortex. 7. Determine cell number by e.g. using Trypan blue exclusion or a cell counter. Seeding of upcyte Hepatocytes for cellbased assays in a 96-well plate 1. Dilute upcyte Hepatocytes in pre-warmed High Performance Medium to a cell density of cells/ml. The optimal seeding density range is listed in table 1 for the respective culture format. 2. Add ml cell suspension per well of a 96-well plate (25,000-50,000 cells/well). Add pre-warmed upcyte Hepatocyte High Performance Medium to a total volume of 200 µl per well. 3. Culture the cells in an incubator maintained at 37 C, under an atmosphere of 95% air/5% CO2 for the desired length of time. For induction studies, we recommend a pre-culture period of up to 7 days. 4. Change the medium every 2-3 days. Page 2 of 6

3 Culture/Treatment period Attachment period Please note: unless indicated otherwise, Medicyte products and services and the Mimetix scaffold are for research purpose only. Do not use for diagnostic or therapeutic applications. Table 1 lists suggested seeding volumes and densities for the different Mimetix scaffold formats. Table 1 Recommended seeding densities for Mimetix Plate format: Surface area (cm 2 ) Cell number per well (in million) 96-well plate well plate well plate well plate with hanging inserts Cancer Cell 2002, Vol 2 (3), pp Thaw the cells according to PFU No. 1. For MIMETIX 12-well plates: Seed x 10 6 cells per well For MIMETIX 96-well plates: Seed x 10 6 cells per well Allow the cells to attach overnight in an incubator Allow the cells to attach overnight in an incubator Change the medium every 2-3 days Check for signs of nutrient depletion (change in color of the phenol red indicator, glucose consumption) Measure endpoint e.g. CYP activities or mrna expression level Multiple endpoints can be measured in the same well Page 3 of 6

4 Results Cell penetration into the scaffold Figure 1 shows a combined fluorescence/ brightfield image of upcyte Hepatocytes growing in the Mimetix scaffold for 5 days after seeding. A 12-well plate with removable scaffold discs (4 µm fibre diameter, 50 μm thickness) was used for this experiment. A scaffold thickness of 50 µm supports the growth of around three layers of cells (see cross sections on the upper and right edge) which demonstrate 3D morphology; yet is translucent enough for imaging with fluorescence-based microscopy. Figure 2 Cell viability and proliferation of upcyte Hepatocytes in Mimetix over 7 days Basal CYP3A4 activity in Mimetix over 12 days Figure 3 shows the CYP3A4 activities of upcyte Hepatocytes in the Mimetix scaffold over 12 days in culture. Figure 1 Fluorescent/brightfield confocal image of upcyte Hepatocytes in Mimetix (nuclei in blue). Cell viability over 7 days: Mimetix vs. 2D Figure 2 highlights the proliferation of upcyte Hepatocytes (50,000 cells per well) within the Mimetix scaffold (96-well format, 4 µm fibre diameter, 50 μm thickness) and a standard 2D 96-well plate. The Mimetix scaffold provides an extra cellular matrix-like environment, which supports the growth of upcyte Hepatocytes. Cell proliferation in 3D in the Mimetix scaffold is initially slower than in 2D, but continues beyond the point at which cell numbers start to decrease in 2D. Figure 3 CYP3A4 activity of upcyte Hepatocytes in Mimetix over 12 days (expressed as mean ± SD in 3 wells) Activities increased over time and reached a maximum at day 10. A 6-well plate with hanging inserts (4 μm fibre diameter, 100 μm thickness) was used for this experiment. CYP3A4 activity was normalised against the amount of protein, which was calculated according to the initial seeding Page 4 of 6

5 volume (1 million upcyte Hepatocytes contain approx. 1 mg of protein). Basal/induced CYP3A4 activity in Mimetix vs. 2D The CYP3A4 enzyme activity of upcyte Hepatocytes was studied after 7 days of culture and subsequent induction with rifampicin for 3 days within a Mimetix 96-well plate (4 µm fibre diameter, 50 μm thickness) and a standard 2D 96-well plate. upcyte Hepatocytes derived from two different donors were used in this study. Those of donor are more optimised for CYP expression in 2D than those of donor 151. The basal CYP3A4 activities of donor 151 are about 10 times lower than those of donor Figure 4a shows that for both donors, CYP3A4 activity of upcyte Hepatocytes can be induced in Mimetix as well as in 2D; i.e. by about 3 times for donor and 5-9 times for donor 151. Induced levels were between pmol/mg/min (donor ) and pmol/mg/min (donor 151). In the case of upcyte Hepatocytes derived from donor , whose proliferation capacity in 2D has been increased through upcyting to a significant extent, there was no significant difference in basal or induced CYP3A4 activity in 2D vs. Mimetix. However, upcyte Hepatocytes derived from donor 151 exhibited higher basal and induced activities of CYP3A4 in Mimetix than when cultured in a 2D monolayer. This suggests that cells that are closer to the primary phenotype (i.e. not optimised for 2D culture) benefit more from being in a 3D environment, such as the Mimetix scaffold, than those optimised for growth in 2D. Figure 4 Basal and induced CYP3A4 enzyme activities of upcytehepatocytes in Mimetix vs. 2D. a) Donor , b) Donor 15 Page 5 of 6

6 Product Supplements/Components Product number upcyte Hepatocyte Thawing Medium upcyte Hepatocyte High Performance Medium ready-to-use ready-to-use (100mL) Animal derived component free (500mL) Supplement A (500mL) L-glutamine (not in Kit) UH0-MT0-050 UH0-MT UH0-ME UH0-MEf-K500 UH0-ME0-K500 MIMETIX scaffolds ready-to-use 000-Pmi-ip Pmi-p Pmi-p Pmi-p96 Purchaser Notification Limited Use Label License (The Electrospinnin g Company) The electrospun scaffold (Mimetix) is provided under an intellectual property license from The Electrospinning Company. The transfer of the Mimetix scaffold is conditioned on the buyer using the purchased product solely in research conducted by the buyer, and the buyer must not use, sell or otherwise transfer this product or its components for: (a) diagnostic, therapeutic or prophylactic purposes; or (b) resale, whether or not resold for use in research; or (c) for the commercial production of therapeutic, diagnostic, or prophylactic products or other products not intended for research use; or (d) to provide a service to deliver information or materials to a customer other than for research purposes of such customer. For information on purchasing a license to this product for purposes other than research, contact The Electrospinning Company, R70 Rutherford Appleton Laboratory, Harwell Oxford, Didcot OX11 0QX, UK or info@electrospinning.co.uk. Limited Use Label License (Medicyte) The cellular product (upcyte cells) generated using the upcyte technology is provided under an intellectual property license from Medicyte GmbH. The transfer of the upcyte cells is conditioned on the buyer using the purchased product solely in research conducted by the buyer, and the buyer must not use, sell or otherwise transfer this product or its components for: (a) diagnostic, therapeutic or prophylactic purposes; or (b) resale, whether or not resold for use in research; or (c) for the commercial production of therapeutic, diagnostic, or prophylactic products or other products not intended for research use; or (d) to provide a service to deliver information or materials to a customer other than for research purposes of such customer. For information on purchasing a license to this Medicyte product for purposes other than research, contact Medicyte GmbH, Heidelberg, Germany or sales@medicyte.com Limited Use Label License (Life Technologies Corporation) During the generation of these upcyte cells a product licensed from Life Technologies Corporation containing the following Limited Use Label License was used: This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, and the buyer must not use, sell or otherwise transfer this product or its components for: (a) diagnostic, therapeutic or prophylactic purposes; or (b) resale, whether or not resold for use in research; or (c) for the commercial production of therapeutic, diagnostic, or prophylactic products or other products not intended for research use; or (d) to provide a service to deliver information or materials to a customer other than for research purposes of such customer. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA USA or outlicensing@lifetech.com. Page 6 of 6

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