Have a question about one of our products? Check out our technical library for recently asked questions from other scientists around the world.
Technical Library
Recent Entries in Technical Library
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I would like to know the number of population doublings for PromoCell mononuclear cells (C-12907/C-12901) when grown in Mononuclear Cell Medium (C-28030).
Mononuclear cells are mostly used in immunology, infection biology, hematology and cancer research to study subpopulations of blood cells.
Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell.
Please note: Depending on the conditions, the ratio of the subpopulations will gradually change, as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells, endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity, viability or metabolism).Related Links and Documents
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How can I calculate the population doubling time?
The population doubling time or generation time (tg) is usually calculated during the logarithmic phase of growth. It specifies the time (t) in hours needed by the culture to double its cell number.
tg = t / nn: number of population doublings
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What is the precise localization for a) HNEpC, b) HTEpC, c) HBEpC, and d) HSAEpC?
a) HNEpC (Human Nasal Epithelial Cells) are isolated from nasal mucosa
b) HTEpC (Human Tracheal Epithelial Cells) from the surface epithelium of trachea
c) HBEpC from the surface epithelium of bronchie, and
d) HSAEpC (Human Small Airway Epithelial Cells) from the distal portion of the respiratory tract in the 1 mm bronchiole area
(comprising the cells from bronchioli and alveoli).Related Links and Documents
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What is the difference between “primary cells” and “normal cells”?
Freshly isolated cells that are plated in a tissue culture vessel for the first time are named primary cells or primary culture (corresponding to P0). As soon as they have been subcultured, they should correctly be termed normal cells (> P1).
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Is everything in the PromoCell Skeletal Muscle Cell Differentiation Medium (C-23061) defined?
Yes, our Skeletal Muscle Cell Differentiation Medium is completely defined. The SupplementMix (C-39366) consists of recombinant human insulin.
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How can I be sure that the cells in my melanocyte culture are melanocytes? Have I understood it correctly that the medium is selective for melanocytes?
After isolation and purification of our primary human melanocytes, the cells are checked during quality control whether they show a typical morphology and whether they express the marker Mel-5. Mel-5 is a 75 kDa glycoprotein usually expressed by normal melanocytes.
Our Melanocyte Growth Medium (C-24010) has been developed to promote melanocyte growth in vitro. It does not however, completely block the growth of other cells (such as: NHEK or NHDF). Therefore, it is important to have a pure melanocyte culture from the very beginning.
In contrast, the Melanocyte Growth Medium M2 (C-24300) and Medium M3 (C-24310) are much more selective and repress the growth of contaminating cells much better.Related Links and Documents
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For how long can the cardiac myocytes (C-12810) be grown in culture?
PromoCell guarantees 15 population doublings (PDs) if the HCM are grown in Myocyte Growth Medium. Depending on the cell lot and the culture conditions, the cells can be maintained in culture for > 6-8 passages corresponding to a period of 1-2 months.
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Did PromoCell try plating the cryo-macrophages in 96-well plates?
We did not plate the cryo-macrophages in 96-well plates. However, we heard from other customers that they have successfully used our macrophages in this kind of plate.
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Will my NHEK (isolated in PromoCell Keratinocyte Growth Medium 2) grow in PromoCell Keratinocyte Growth Medium 3?
Yes, the NHEK-GM2 (C-12001, C-12003, C-12005, C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals, they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs.
Conversely, NHEK-GM3 (C-12011, C-12013, C-12015, C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% – 90%), they grow slightly slower compared to Keratinocyte GM3, but also reach > 15 PDs.Related Links and Documents
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For which cytokeratin are the keratinocytes tested?
PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.
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Can I thaw a vial of HBEpC and directly seed it on transwells for my ALI-experiment?
Yes, but remember to thaw and seed the cells in our growth factor containing Airway Epithelial Cell Growth Medium as the cells need to expand and proliferate for some days.Always use a seeding density of 150.000 cells/cm2, even if you plate the cells on transwells directly after thawing. Do not forget to coat the inserts with 30 µg/ml Collagen Type I solution (e.g. Corning Inc®., product number 354236) before you seed the cells.
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What could be the reason for a lower yield when using M2 Macrophage Generation Medium compared to M1 Macrophage Generation Medium for a culture started with the same monocytes?
For the M2 Macrophage Generation Medium, it is extremely important that the shelf life of 2 weeks (after addition of the cytokines) is not exceeded; the yield will quickly decrease thereafter. It is best to use the M2 medium as fresh as possible to avoid discrepancies between M1 and M2 yield.
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Can PromoCell recommend a medium to co-culture endothelial cells and fibroblasts?
Some of our customers have successfully used PromoCell Endothelial Cell Growth Medium MV2 (C-22022) when co-culturing Human Coronary Artery Endothelial Cells (HCAEC; C-12221) and Normal Human Dermal Fibroblasts (NHDF; C-12300).
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What are the advantages of using PromoCell DC Generation Medium XF for dendritic cell generation?
Our DC Generation Medium XF (C-28052) has a xeno-free formulation. It provides a complete media system (ready-to-use, all cytokines included) and shows efficient and reproducible in vitro maturation of moDCs from freshly isolated peripheral blood monocytes.
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How does PromoCell ensure that there is no fibroblast contamination in the pericytes? Are the fibroblasts removed by separation with anti-CD90 labeled magnetic beads?
No, we don’t perform CD90 immunomagnetic separation with our pericytes. We can however exclude fibroblast contamination as follows:
1) The presence of fibroblasts in our pericyte cultures would be detectable shortly after cell isolation, as pericytes need up to 2 weeks before they start proliferating. Fibroblasts on the other hand would proliferate immediately and overgrow the culture.
2) Characterization of the isolated pericytes during QC includes flow cytometry of CD146. Pericytes express CD146 whereas placental fibroblasts don’t.Related Links and Documents
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My cells didn’t detach during subculture. What could be the reason?
Please find attached a trouble shooting guide to identify possible reasons for poor detachment during subculture.
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Why is the growth rate of PromoCell’s HDMEC significantly reduced when I remove hydrocortisone from the Endothelial Cell Growth Medium MV Kit (C-22120)?
Epidermal Growth Factor (EGF) and hydrocortisone have been reported to have a synergistic effect on the growth of microvascular endothelial cells. Accordingly, if you remove the hydrocortisone, the proliferation of HDMEC is clearly affected.
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Should I warm the trypsin prior to use?
We recommend to use the trypsin as well as the other detach solutions at room temperature to avoid overtrypsinization and irreversible cellular damage.
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Can I use my own culture medium or other commercially available media for culturing PromoCell Normal Human Cells?
Our Normal Human Cells have been cultured and tested in our growth media and have adapted to these conditions. Using other media may yield unsatisfactory results due to suboptimal supplies of nutrients and growth factors. PromoCell can only guarantee good cell growth (as stated in the Certificate of Analysis), when the cells are grown in the recommended media.
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Does the differentiation capacity of Human Mesenchymal Stem Cells change over time? When should I best induce differentiation?
We have tested the differentiation capacity of our hMSC into adipocytes, chondrocytes and osteoblasts over time and still see good differentiation rates after 10 population doublings, i.e., at passage 5. However, the differentiation potential declines with ongoing population doublings. To obtain optimal differentiation rates, experiments should be performed as early in culture as possible.
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How many cells do the cell pellets contain?
PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.
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What are the characteristic markers of HDLEC (C-12216, C-12217) and HDBEC (C-12211, C-12225)?
- HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
- HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.
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How often should I change the growth medium when culturing PromoCell Normal Human Cells?
Generally, the medium should be changed every 2-3 days.
Please note: Following thawing, the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.
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What would be the likely impact of using PromoCell Fibroblast Growth Medium (C-23010) instead of DMEM + 10% FCS for the growth of juvenile fibroblasts (C-12300)?
PromoCell Fibroblast Growth Medium is serum-free and therefore not subjected to the lot-to-lot variations observed with DMEM/10% FCS. Our Fibroblast Growth Medium therefore allows for much more standardized cell culture conditions.
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Can PromoCell supply melanocytes from asian donors?
The majority of our skin tissue donors are caucasians. But occasionally we also get skin biopsies from asian and black donors.
Please contact our Technical Customer Support if you need cells from a particular phototype or origin. They will check our inventory and send you a list of available cell lots.Related Links and Documents
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How many bottles of medium will I need to grow 1 vial of HUVEC out to 15 population doublings?
The amount of media needed per vial depends on the growth characteristics of the cells, the size of the TC vessels and the split ratios used, the frequency of media changes, the type of experiments you perform, etc. It is therefore difficult to give definite quantities. As a rough guideline, 1-2 bottles (500 ml each) are needed for 1 vial of HUVEC.
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Which cytokine concentrations does PromoCell recommend for activating M1/M2 macrophages?
You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.
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What is the difference between PromoCell NHEK and NHEK GM3?
The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011), the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both, NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.
Related Links and Documents
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Can you drive organoid orientation (inward vs outward)?
Yes. The orientation can be triggered by the use or lack thereof of ECM.
Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/, where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM.
* Please note that we have not tested this method in our labs and thus cannot guarantee it.
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What are raw materials or ancillary materials and how do these differ from pharmaceutical excipients?
Raw materials come in contact with the cell or tissue product during manufacturing but are not intended to be part of the final product. For example, cell culture media and reagents can be used in research and production of cell-based drugs and therapies. In this case they are defined as raw materials. During drug, cosmetic, and cell therapy development, the quality of raw materials must be carefully considered as they may have an effect on the efficacy of the final product and subsequent safety for the patient [1].
The nomenclature for raw materials differs between the regions. The terminology “raw material” is used by European regulators. In other regions the synonymous term ancillary material is used.
Raw materials or ancillary materials are commonly labeled as “not for use in clinical or diagnostic procedures” or “for ex vivo use only and not intended for human in vivo applications”.
Pharmaceutical excipients are substances that are included in a pharmaceutical dosage form not for their direct therapeutic action, but to aid the manufacturing process, to protect, support or enhance stability, or for bioavailability or patient acceptability. They may further assist in the effectiveness and/or delivery of the drug in use and maintain the integrity of the drug product during storage.
[1] Solomon J, Csontos L, Clarke D, Bonyhadi M, Zylberberg C, McNiece I, Kurtzberg J, Bell R, Deans R. Current perspectives on the use of ancillary materials for the manufacture of cellular therapies. Cytotherapy. 2016 Jan;18(1):1-12.
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After detaching the macrophages for flow cytometric analysis, I noticed that the washing and centrifugation steps take a long time. Can I shorten the spin time to 10 minutes at 350 x g?
Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented, but intact, macrophages.
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We are culturing human coronary artery smooth muscle cells (HCASMC) from your company. Are these cells obtained from large arteries like LAD or left Circumflex or from small branches of LAD etc. ?
Our HCASMC (C-12221) are isolated from the large arteries, i.e. from
- Right coronary artery
- Left main coronary artery
- Circumflex coronary artery and
- Left anterior descending coronary artery.
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How long does it take to differentiate monocytes into mature DCs using PromoCell DC Generation Medium/DC Generation Medium XF?
It takes 7-8 days to generate fully mature myeloid dendritic cells.
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What is the exact localization of PromoCell’s Human Pericytes? How many doublings can they perform in culture?
Our hPC-PL (C-12980) are isolated from microvessels of the human placenta, from the chorionic villi.
The number of populations doublings is not determined for each individual cell lot, but in our experience, they can be grown for at least 15 population doublings.Related Links and Documents
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My Normal Human Cells didn’t attach after thawing. What could be the reason?
Poor attachment after thawing can be a result of inappropriate freezing, storing or thawing the cells as well as from inadequate culture conditions (medium, incubator). The attached trouble shooting guide should help you to identify the possible reasons.
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Is it possible to split differentiated adipocytes in order to perform more experiments?
Generally, you can split differentiated adipocytes. But these cells lose their ability to proliferate after differentiation and you can’t expand the culture any more. Therefore it is recommended to plate the cells into the needed vessels (e.g. multiwell plates) prior to induction of differentiation so that trypsinization isn’t necessary.
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What is the concentration of the trypsin inhibitor in TNS?
Our TNS solution contains 0.05% (w/v) trypsin inhibitor from soy bean in HepesBSS/0.1% BSA.
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How do mesenchymal stem cells from different tissues differ in terms of their biological function?
The human MSC derived from bone marrow, adipose tissue, and umbilical cord matrix are from different origins, but with comparable biological properties and function. Depending on the tissue of origin, they may have a higher preference for differentiation into one particular cell type and a lower preference for another one, but they all still retain the differentiation potential for the mesenchymal lineage.
- MSC-BM: very good differentiation into bone cells, chondrocytes & fat cells
- MSC-UC: very good differentiation into chondrocytes; but weaker potential into fat/bone cells
- MSC-AT: very good differentiation into fat cells
Related Links and Documents
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Which plates does PromoCell recommend as 96-well U bottom suspension plates for chondrogenic differentiation of Human Mesenchymal Stem Cells?
For chrondrogenic differentiation it is important that the cells do not adhere to the wells. During differentiation, the cells form spheroids which float in the medium. Therefore, there are no special requirements for the wells as long as they are U bottom shaped and suitable for suspension culture.
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At what passage do PromoCell supply the cell pellets?
The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells.
Examples:
HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2.
SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3.
In contrast, our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step. -
From what tissue do PromoCell isolate their HUtMEC (C-12295)?
Our HUtMEC are isolated from the middle layer of the uterine wall (myometrium). The tissue donors were not pre-treated with hormones.
Related Links and Documents
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What is the composition of the Osteoblast Supplement (C-39615)?
Our Osteoblast Supplement consists of FCS (10 % [v/v] final concentration) which is specifically tested to support optimal growth of Normal Human Osteoblasts.
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Should the skeletal muscle cells be split after adding the Differentiation Medium (C-23061)?
After adding the Skeletal Muscle Cell Differentiation Medium, myoblasts will start to differentiate into myotubes and stop growing. The cells should not be split anymore. It is recommended rather to plate the SkMC into the needed vessels, (e.g. multiwell plates), prior to induction of differentiation and to perform studies directly on differentiated cells. If it is necessary for your tests to detach the myotubes and they are difficult to trypsinze, you can use a “rubber policeman”.
Related Links and Documents
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Does PromoCell offer a special 3-D system to block de-differentiation or does PromoCell have special culture conditions that stabilize the chondrocyte phenotype?
PromoCell Chondrocytes (HCH) can be expanded in normal monolayer culture using our Chondrocyte Growth Medium (C-27101). The Medium consists of an optimized formulation and is supplemented with 10 % FCS. De-differentiation of chondrocytes is a known phenomenon observed during in vitro-culture after a period of approx. 2 weeks, but in vitro-culture is needed to expand the cells.
Once a suitable cell number is obtained, the monolayer system can be changed to a more complex 3-D system either by culturing the cells on substrates like alginate beads, gels, or degradable polymer scaffolds or by using 3-D spheroid culture. Using appropriate conditions, re-differentiation is triggered and the cells start producing cartilage-specific ECM again.
PromoCell does not supply a special culture system but we use 3-D spheroid culture and Alcian Blue staining to characterize our chondrocytes during quality control. The protocol is very similar to the one we use for chondrogenic differentiation of MSC.Related Links and Documents
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Does the PromoCell 3D Tumorsphere Medium XF (C-28070) maintain the stem cell character of cancer stem cells?
During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.
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Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?
No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
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Can the Cytokine Mix E for the Hematopoietic Progenitor Expansion Medium XF (C-39891; 5 ml) be safely aliquoted?
Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.
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Can you control size / size distribution of organoids?
Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/
For best results we recommend the following:
- Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
- For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.
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What is the EXCiPACT™ GMP certification?
Pharmaceutical excipient production should be carried out in accordance with the principles defined in the Good Manufacturing Practices (GMP). With that in mind, a group of industry experts including EFCG, IPEC and PQG worked together to develop the EXCiPACT™ certification scheme for pharmaceutical excipient suppliers.
EXCiPACT™ is a non-profit organization that oversees and operates an independent, high-quality, third-party certification system. This certification system is available to pharmaceutical manufacturers and distributors around the world.
To achieve EXCiPACT™ GMP certification, we demonstrated that our pharmaceutical excipients are manufactured according to the GMP guidelines, as well as ensuring compliance in our raw material supply chain, production environment, production processes and storage conditions.
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