LSP-DOC-3339 (ID 11862) Human Lysate FAQs.docx

Frequently Asked Questions (FAQs): Human Platelet Lysate What is hPL? Human Platelet Lysate (hPL) is a growth supplement obtained from human . hPL contains abundant human growth factors including PDGF, bFGF, EGF, TGF-beta1, VEGF, other cytokines and proteins which yield superior results when culturing many types of human cells, particularly human MSC, regardless of their tissue source. Serum (traditionally FBS or Human) is the supernatant of clotted blood, hPL is manufactured from lysed platelets. How is it collected? hPL is derived from human donor platelets collected from healthy consented volunteer donors at certified blood banks. All individual donors and complete batches are fully tested for pathogens according to 21CFR610. What are the benefits to using hPL instead of FBS? Performance: The future of cell therapy is to be xenogeneic-free and therefore human platelet lysate offers xenogeneic free/serum substitutes for replacing FBS and improving cell culture performance. Cost: FBS is commonly used at concentration between 10-20% in cell culture. However due to the high performance effects of human platelet lysate it is typically used at lower concentrations; 2.5%, 5% or 10%. Batch-to-batch variation: Batch-to-batch variability of hPL is significantly lower than that of FBS because it is derived from standardized human blood products. Since hPL is obtained from multiple donors units that are subsequently pooled in large batch sizes, it enables the manufacture of a consistent product with minimal lot-to-lot variation. What concentration of hPL should I use? Human Platelet Lysate (hPL) can be used at a variety of concentrations, from 10% in basal media to as little as 2.5%. The table below indicates the concentrations of hPL used for a variety of different cell types and applications. Which cell types has hPL been shown to work with?

Concentration Species Cell Type Cell Name Description Used (%) Bovine Primary Cell CEC Corneal Endothelial Cells 2.5 - 10% Hamster Cell Line CHO Chinese Hamster Ovary epithelial Unknown Human Cell Line HEK-293 Human Embryonic Kidney 293 cells 1 - 10% Human Cell Line HeLa HeLa Cervical cancer cells 10% Human Cell Line HFFF2 HFFF2 Dermal fibroblasts 5 - 20% Human Cell Line HL-60 Human leukaemia cell lines 20% Human Cell Line HUVEC Human umbilical vein endothelial cells 5 - 20%

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Human Cell Line JURKAT Human leukaemia cell lines 10% Human Cell Line K562 Myelogenous leukaemia 10% Human Cell Line KG-1 Human leukaemia cell lines 20% Human Cell Line MCF-7 Human continuous tumour 10% Human Cell Line NCTC 2544 Dermal keratinocytes 5% Human Cell Line NCTC 2544 Dermal keratinocytes 5 - 20% Human Cell Line A-549 Human continuous tumour 5% Human Cell Line Caco-2 Human continuous tumour 5% Human Cell Line HaCaT Keratinocyte cell line from adult human skin 5% Human Cell Line HEK-293 Human Embryonic Kidney 293 cells Unknown Human Cell Line HGF-1 Human Gingiva Fibroblasts 5% Human Cell Line MCF-7 Human continuous tumour 10% Human Cell Line U-251 MG Human continuous tumour 5% Mouse Cell Line C2C12 Myoblastoma Cells 1 - 20% MMT Mouse Cell Line Mouse mammary tumour Unknown 060562 Mouse Cell Line Neuro-2a Neuroblastoma cell line Unknown Mouse Cell Line NIH/3T3 Mouse Embryonic Fibroblasts 1 - 10% Mouse Cell Line RAG Mouse adenocarcinoma cell line Unknown Rabbit Cell Line SIRC Statens Seruminstitut Rabbit Cornea 1 - 10% Rat Primary Cell MSC Mesenchymal Stem Cells 2.5 - 5% Rat Cell Line R2C Testis from rat Unknown The above list represents cell lines and conditions that have been stated in published literature or from customer testimonials. We would recommend testing your cells with a titration range to determine the ideal conditions for your particular cells and application. What does it mean when hPL is labelled as Research Grade, GMP or GMP Clinical Grade? Research Grade material is manufactured to meet the needs of basic research and development. This material supports the in vitro propagation and maintenance of various human cell types. In addition, it provides a cost effective alternative to using serum-free media. Material is supplied in validated PETG NALEGENE bottles (25 mL, 100 mL and 500 mL). GMP Grade material is manufactured under GMP-controlled conditions for use in GMP compliant cell culture SOPs. This material is provided in cryo-bags (100 mL) or in validated PETG Nalgene bottles (100 mL and 500 mL). GMP Clinical Grade material is manufactured and gamma irradiated under GMP conditions to comply with the high safety guidelines for clinical applications. Due to pathogen-reduction, GMP Clinical Grade hPL provides a high safety level for expansion of different human cell types (particularly adult stem cells, e.g. MSCs) for clinical and therapeutic applications.

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Which hPL product should I choose? Product Key Benefits Pack sizes Fibrinogen Requires Animal- available -depleted heparin? component free Human Platelet Animal serum-free; 25 mL, 100 mL No Yes Yes* Lysate Solution most cost effective & 500 mL option

Human Platelet Animal serum-free; 10 mL, 50 mL, Yes No No** Lysate -FD fibrinogen-depleted 100 mL & 500 mL

Human Platelet Animal component- 10 mL, 50 mL, Yes No YesƗ Lysate-XF free; fibrinogen- 100mL & 500 depleted mL

Human Platelet Animal serum-free; Customisable No Yes Yes* Lysate - Customised customisable; small scale process development

*does not contain any heparins (these are added at the point of use). **contains residues of porcine- derived heparin. ᵻ contains residues of fully synthetic heparin. Which Heparin product should I use? Only the Human Platelet Lysate Standard and Customised products requires the addition of heparin. There are two types of heparin available: 1. Heparin - Standard animal-derived heparin (5,000 U/mL) - HSUP-001M. 1. Xeno-Free Heparin - Animal component-free heparin, fully synthetic and preservative-free (12.5 mg/mL) - HSUP-XF-001M. What does ‘Xeno-Free’ mean? Our ‘Xeno-Free’ products are non-xenogeneic and animal serum-free. i.e. they do not contain material from species other than human origin. Materials that are described as being ‘Xenogenic’ are derived from a different species and therefore are not genetically or immunologically compatible. This is very important for cell therapy and clinical applications. What is the difference between ‘animal-serum free’ and ‘animal-component free’? All of our hPL products are derived from human platelets and therefore are naturally free form other animal derived material. No animal serum is added during the manufacture of these products.

For a product to be animal-component free, no products can be added to the product during the manufacturing process that are derived from another species. For an animal-component free option (i.e. Page 3 of 8 Life Science Production, a Division of Life Science Group Ltd. Tel: +44 (0) 1234 889180; Email: [email protected] Web: www.lifesciencegroup.co.uk LSP-DOC-3339 (ID 11862) Human Platelet Lysate FAQs.docx

free from non-human species), either choose Human Platelet Lysate Standard with Xeno-free heparin, or Human Platelet Lysate Solution XF (which is manufactured using Xeno-free heparin, a fully synthetic heparin product). What does it mean when hPL product is described as being Fibrinogen-depleted? For both Human Platelet Lysate-XF (Xeno-Free) and Human Platelet Lysate-FD (Fibrinogen Depleted), a fibrinogen depletion step is performed during the manufacturing process in order to remove all fibrinogen. In addition, heparin is added during the manufacturing process. This means that you do not need to add heparin while preparing the cell culture medium. For Human Platelet Lysate FD, the heparin used in manufacturing is derived from porcine intestine (and therefore not xeno-free). For Human Platelet Lysate XF, the heparin used is a xeno-free version, and is fully synthetic, offering a completely xeno-free (animal-component free) end product. Why is heparin not required for the FD and XF products? Heparin is an anti-coagulant that prevents the fibrinogen in human platelet lysate from clotting in the cell culture medium during cell expansion. Both Human Platelet Lysate XF and Human Platelet Lysate FD are fibrinogen-depleted and so do not require the addition of heparin in the cell culture media. What cell types are particularly recommended? Human Mesenchymal Stromal Cells from Bone Marrow (hMSC-BM) Human Mesenchymal Stromal Cells from Umbilical Cord (hMSC-UC) Human Mesenchymal Stromal Cells from Adipose Tissue (hMSC-AT) Human Dermal Fibroblasts (HDF) In addition, see FAQ ‘Which cell types has hPL been shown to work in?’, for a more detailed table of other cell types that hPL has been shown to work with. Can I use hPL in diagnostic or therapeutic applications? No. hPL is for in vitro experimental and research use only (RUO) and the product is not intended for human or animal diagnostic or therapeutic uses. What tests are carried out on hPL? Each batch of hPL is tested to ensure the absence of bacteria, fungi, yeast, mycoplasma (M. fermentans, M. arginini, M. orale, M. hyorhinis, M. salivarium, M. hominis, M. pneumoniae, Acholeplasma laidlawii, M. synoviae and Ureaplasma species). Batches are also tested for the ability to support growth of specific cell lines. In addition, each batch is also tested for standard parameters. These include endotoxin, pH, osmolality and total protein. Details for each batch are supplied on the Certificate of Analysis (COA). Which viral infections are batches of hPL tested for? Our hPL products are manufactured from platelet units obtained from healthy blood donors who have been tested and found negative for HBsAg, HIV I/II antibody, CMV, HCV- and HBC-Antibody, HIV-, HCV-,

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HBV-, HAV-, PVB19-NAT, Treponema pallidum and Syphilis.

Is it sterile? hPL is aseptically processed. Each batch of hPL is tested for the absence of bacteria, fungi, yeast, and mycoplasma. Batches of hPL are also sterile filtered. Results of microbe testing of each batch are supplied in the COA. However, please note that sterility is not guaranteed after opening. The use of aseptic techniques during handling is recommended. A gamma-irradiated option (GMP-Clinical grade) for the purpose of virus-inactivation is available for all hPL products. NOTE: our gamma-irradiated products are manufactured in the US. What do the different formats mean? Human Platelet Lysate Standard – requires the addition of heparin to the final cell culture medium. Choose from a standard animal derived heparin (porcine), or a xeno-free heparin (fully synthetic and preservative free). For human ex-vivo tissue and cell culture processing applications. Human Platelet Lysate FD (Fibrinogen Depleted) – A fibrinogen-depleted hPL that does not require the addition of heparin. However, it may contain residues of heparin. For human ex-vivo tissue and cell culture processing applications. Human Platelet Lysate XF (Xeno-Free) – A completely xeno-free fibrinogen-depleted hPL. Does not require the addition of heparin, however it may contain residues of synthetic, xeno-free heparin. For human ex-vivo tissue and cell culture processing applications. Human Platelet Lysate-Customized – A custom-manufactured human platelet lysate solution that requires the addition of heparin to the final cell culture medium. Designed for the small-scale process development needs of life cycles, protein therapeutics, biologics, vaccine expression or small-volume testing prior to GMP scale-up. What pack sizes are available for hPL? Research Grade: 25 mL, 100 mL and 500 mL Nalgene bottles. GMP Grade: 100 mL cryo-bags and 100 mL Nalgene bottles and 500 mL Nalgene bottles. GMP-Clinical grade (gamma irradiated): 50 mL, 100 mL and 500 mL Nalgene bottles. Can I sample batches of hPL? Yes. Samples of hPL are available for testing prior to selection of a suitable batch. Typical sample size is 25 mL and reservations are held for a period of four weeks, pending evaluation. What is the shelf life for hPL? hPL has a shelf life of 24 months from the date of manufacture, provided they are stored appropriately. We would recommend enquiring about the shelf life of each available batch if it is important to have a long shelf life following purchase.

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What is the recommended storage and handling for hPL? The recommended storage is -20°C or below. hPL products can be stored long term at -80°C. Thaw frozen hPL product overnight at 4°C or for 1 hour in a 37°C water bath before use. Once hPL product is thawed, it is recommended to fully use it or to aliquot and re-freeze samples of unused hPL at -20°C. Completed medium preparation (e.g. 5% hPL) can be stored at 4°C and is stable for approximately four weeks. It is recommended to avoid multiple freeze-thaw cycles as this can lead to an increase in the formation of insoluble particles. All biological material should be handled as potentially infectious. It is essential that universal precautions should be employed when handling hPL. Are there any specific precautions required when using hPL? Despite all testing, proper safety precautions for potentially infectious agents must be taken. All human blood products should be handled in accordance with currently acceptable bio-safety practices and guidelines for the prevention of blood borne viral infections. How is hPL shipped? hPL will be shipped frozen, by overnight courier in polystyrene moulded boxes with dry ice as this will ensure that the material arrives frozen in good condition. How is the Heparin shipped? Both Heparin and Xeno-Free Heparin are shipped at ambient temperature. Store at +4°C on arrival. What should I do if I get precipitation in my hPL? The presence of particulate matter is a result of fibrinogen converting to insoluble fibrin resulting from freezing and thawing human platelet lysate. This particulate matter does not indicate contamination of any sort, and does not have any negative impact on cell culture performance. To avoid particulate matter from forming, we suggest minimal freeze / thaw cycles when using our products. Should insoluble particles appear in thawed hPL we recommend removing particles by centrifuging at 3,400 x g for 3-5 minutes. If clotting or insoluble particles appear in the final medium, we recommend filtering the complete culture medium after diluting in the basal medium to remove insoluble particles. Filtering the completed medium (e.g. 5% hPL), will not affect supplemented cell culture performance. However, filtering is NOT recommended for 100% concentrate hPL as this may reduce cell culture performance. Is there a difference between the actual manufacturing and formulation of the Research Grade hPL and the GMP Grade hPL aside from the extra testing and documentation that the GMP hPL undergoes?

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Technically and procedurally there is no any difference between our research and GMP Grade products. The difference is in the additional documentation supplied for the manufacturing process of the GMP Grade material. Do you see a difference in cellular performance between the Research Grade and the GMP Grade hPL? We have not seen any difference in performance since these are identical products manufactured to the same SOP. Our customers also report that there is no difference on performance levels between the research and GMP grade products. Do you see a difference in the performance of your Human Platelet Lysate that is xeno-free from your Human Platelet Lysate that requires the addition of heparin? Based on our testing we can see minimal differences between both products in terms of performance. The xeno-free products were developed to provide the same levels of performance but in a xeno-free environment. How do I use hPL? 1. Thaw Human Platelet Lysate Solution overnight at 4°C or for 1 hour in a 37°C water bath. 2. Prepare complete MSC culture medium by adding Human Platelet Lysate Solution to basal medium (i.e. Dulbecco’s Modified Eagles Medium-Low Glucose; DMEM-LG) with 2 mM L-glutamine and 100 U/mL Penicillin/streptomycin as final concentration. 3. hPL shows optimal growth of MSC at 5% (v/v). However, for higher cell proliferation rates, it is recommended to use 10% (v/v) hPL. 4. We recommend seeding MSCs at approximately 3x103 ~ 6x103 per cm2. 5. For Human Platelet Lysate Standard ONLY, to avoid coagulation of the complete MSC culture medium, heparin should be added at a final concentration of 2 U/mL Heparin (cat.no. HSUP-001M) or 0.024 mg/mL Xeno-Free Heparin (cat. no. HSUPXF-001M). NOTE: Human Platelet Lysate FD and Human Platelet Lysate XF DO NOT require the addition of heparin, as they are fibrinogen-depleted products. 6. Complete MSC culture medium can be stored at 4°C and is stable for approximately four weeks. How does hPL perform as an alternative to Human AB serum?

From a technical viewpoint, the application of hPL, Human serum (HS) and FBS are very similar. hPL differs to sera products due to the increased volume of cytokines and growth factors contained within the product affecting the performance levels. We would recommend the following paper by Bieback et al. 2009 that demonstrates the comparative performance difference between hPL, HS and FBS.

Bieback, K., et al. (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cell, 27(9):2331-2341.

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How does hPL perform as an alternative to Human Platelet Releasates?

The difference between human platelet releasates (hPR) and human platelet lysate (hPL) that in hPR platelets are activated by thrombin addition before disruption of the platelets. This means that the hPR will only contain those factors released after platelet activation. hPL, in contrast, will contain all of the factors contained within the platelets. Describe the manufacturing process for hPL

All platelets sourced for the manufacture of our hPL are derived from certified centres and comply with all regulations and guideline regarding traceability, viral testing and components added.

Certain products are manufactured under GMP conditions using Grade A/ISO 5 Isolators within Grade C/ISO 7 rooms in compliance with current regulatory requirements. A confirmatory Certificate of Analysis is provided with our GMP products.

Our production process is based on a patented freeze-thaw method to release the growth factors from the platelets. We do not perform any platelet activation using thrombin.

Does hPL require handling in a Class 2 Biological Safety environment?

It is recommended that all biological materials are handled at Biological Safety Level 2.

Support Life Science Production is a division of Life Science Group Ltd. Life Science Production is ISIA Traceability Certified Life Science Group Ltd is an ISO 9001:2015 Certified company

To learn more, contact us: Telephone: +44 (0) 1234 889180 Email: [email protected] Website: www.lifescienceproduction.co.uk Address: PO Box 1519, Bedford, United Kingdom

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