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
298 Entries on 6 Pages
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Can I use the 3D Tumorsphere Medium XF for mouse cell lines?
Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.
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Looking at the detachment protocol of PromoCell’s Macrophage Detachment Solution, I observed that we need to add HSA to the PBS. Can I use FBS instead? Also, why are we adding 2 mM EDTA to the PBS?
- FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.
- The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca2+/Mg2+ free PBS.
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What is the difference between DC Generation Medium Ready-to-use (C-28050) and DC Base Medium (C-28053)?
- DC Base Medium (C-28053) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.
- DC Generation Medium (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.
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Do the PromoCell media contain L-glutamine?
Yes, the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don’t add extra L-glutamine as this can be toxic for the cells.
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What’s the difference between trypsin and accutase?
Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn’t require an inactivation step.
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Why are most of the soluble receptors not produced in E. coli?
Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don’t show any biological activity.
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What is the DMSO concentration in Cryo-SFM?
The DMSO concentration in our Cryo-SFM is 10%.
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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
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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.
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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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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.
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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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In the past, we used MSC Growth Medium (C-28010) in our lab. Can I use MSC Growth Medium 2 (C-28009) instead? What is the difference?
MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation).
You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow, adipose tissue, or umbilical cord) at 4,000 cells/cm².Related Links and Documents
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Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD FalconTM.
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What is the difference between DC Generation Medium XF (C-28052) and DC Base Medium XF (C-28054)?
- DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user’s individual needs.
- DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.
Both, Base Medium XF and Generation Medium XF have a xeno-free formulation.
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What’s the difference between PromoCell HUVECs and HUV-EC-C from ATCC?
PromoCell HUVECs are freshly isolated from umbilical veins. They are cryopreserved at the end of primary culture. After revival, they can be propagated for at least 15 doublings and will senesce eventually.
HUV-EC-C from ATCC is a hypodiploid human cell line of endothelial origin (umbilical vein). The modal chromosome number is 45 occurring in 72% of cells counted. The rate of polyploid cells is 15.8%. The cells have a life expectancy of 50 to 60 population doublings. This indicates that the cells are no longer “normal cells” but have undergone some degree of transformation.Related Links and Documents
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Will the hematopoietic progenitor cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) without Cytokine Mix E (C-39890)?
For the expansion of hematopoietic progenitors (CD133+ cells, CD34+ cells), PromoCell has developed Hematopoietic Progenitor Cell Expansion Medium XF (C-28021), a serum-free and xeno-free formulation. The medium must be supplemented with either Cytokine Mix E (C-39890) or with the user’s own mixture of cytokines. Cytokine Mix E is a ready-to-use mix containing recombinant human TPO, SCF, flt3-ligand, and IL-3.
The strong expansion of the progenitor cells in Expansion Medium XF + Cytokine Mix E persists for at least two weeks, resulting in a 200-300x increase of the cell number with only 20-30% differentiated cells.
Note: If starting with CD133+ cells, the CD133 marker is getting lost during this expansion step. The resulting cells are D34+/CD38–/CD133–.Related Links and Documents
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Can mycoplasma contamination be observed with the naked eye?
No, mycoplasma can only be observed through electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the use of PCR-based mycoplasma tests.
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What antibiotics concentration should I add to the PromoCell media?
If antibiotics are deemed necessary the recommended final concentrations are:
100 U/ml penicillin + 100 µg/ml streptomycin or
50 µg/ml gentamicin + 50 ng/ml amphotericin BPlease note: Addition of antibiotics can reduce the growth rate of the cells.
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To what extent will the CD34+ Progenitor Cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021)?
For optimal cell growth, the human CD34+ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO, SCF, flt3-ligand and IL-3. The strong expansion of CD34+ cells will persist for approx. 2 weeks. The expansion factor is usually between 75 and 200-fold.
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What are the criteria for selection or exclusion of peripheral blood donors? Is the peripheral blood that PromoCell use to isolate blood cells (MNC-PB, monocytes) derived from completely healthy donors?
The following criteria are applied for blood donations:
a) Donors with light infection like a cold or cough are excluded for one week
b) Donors with infections with a temperature above 37.9°C and/ or antibiotics therapy are excluded for 4 weeks
c) Blood pressure: exclusion only if systolic blood pressure is < 100 mm Hg or > 180 mm Hg or if diastolic blood pressure is > 100 mm Hg. No exclusion if blood pressure is drug treated and in acceptable range.
d) High cholesterol: no exclusion even though medication is used
e) Diabetes: exclusion if diabetes type I or use of insulin
f) Steroid use: exclusion for 4 weeks after application
g) Cancer: exclusion
h) Other chronic diseases: exclusion depends on the type of disease (for example chronic heart disease, autoimmune disease)Related Links and Documents
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What medium should I use to grow or differentiate PromoCell Human Stem and Blood Cells?
The recommended media for a particular cell type are specified on the respective product page (“Recommended Products”) and can be found in the Manual belonging to the cells.
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From what tissue do PromoCell isolate their HPMEC (C-12281)?
Our HPMEC are isolated from peripheral lung tissue of adult donors.
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How should I thaw the PromoCell normal human cells to obtain a high viability?
General protocol for recovery of anchorage-dependent primary cells:
Remove vial from liquid nitrogen, transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min, until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress.
Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min).
The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.Related Links and Documents
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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”.
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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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Is it possible to carry out the macrophage differentiation in either a 96- or 384-well plate, so long as the volumes are adjusted accordingly? If not, is it possible for cells to be detached and re-plated in the desired culture plate?
a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven’t tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%).
Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g., evaporation of media, dry wells, etc.).Related Links and Documents
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I was wondering if you had any suggestions regarding the plasticware to use for monocyte enrichment/macrophage differentiation.
Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence.
For our M1-/M2 Macrophage Generation Media XF we recommend the Nunc plasticware with Nunclon surface – as not only will the detachment efficiency vary (up to 20%), but also the efficiency of the differentiation process itself may be altered.
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What is the pH of your Endothelial Cell Culture Media MV2?
The pH of our Endothelial Cell Basal Media MV/MV2 is 7.4 ± 0.1.
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What’s the difference between PromoCell HUVEC single donor (C-12200) and HUVEC-pooled (C-12203)?
Our HUVEC single donor (C-12200) are isolated from a single umbilical cord, propagated in primary culture, and frozen down at subconfluency.
For the preparation of HUVEC-pooled (C12203), we simultaneously isolate the cells from 2-4 umbilical cords and grow them in separate tissue culture dishes. The cells are pooled after trypsinization given that their growth rates are comparable.
After thawing, our HUVECs (single donor and pooled) are both in P1. The recommended media are Endothelial Cell Growth Medium (C-22010) or Endothelial Cell Growth Medium 2 (C-22011). With respect to cell growth, HUVEC-pooled tend to have a more heterogeneous morphology with slightly more elongated cells but the doubling times are comparabel for both types (typically 18-36 hrs per doubling).Related Links and Documents
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What is the exact localization of PromoCell’s HAoAF (Human Aortic Adventitial Fibroblasts)?
Our HAoAF (C-12380) are isolated from the Adventitia, the outer layer of the aorta. The cells have been characterized as fibroblasts by the expression of fibroblast-specific CD90.
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At what passage are PromoCell Mesenchymal Stem Cells / Pericytes upon arrival?
Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.
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Do I need additional supplements when working with PromoCell growth media? Do I have to add extra FCS?
After addition of the SupplementMix or SupplementPack to our basal medium, you obtain the complete growth medium. No further supplementation with serum or growth factors is required. Please note: Our media do not contain antibiotics. If you wish to use antibiotics you can add penicillin/streptomycin or gentamicin/amphotericin B at standard concentrations. Addition of antibiotics can however reduce the doubling time of the cells up to 30-40%.
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I am interested in purchasing PromoCell Human Mononuclear Cells (C-12907). Could you please let me know whether you have tested them for platelet contamination? If yes, which markers / procedure have you tried?
During the isolation of our Human Mononuclear Cells (hMNC), we first of all pay attention to thoroughly discard the platelet-containing fraction before we aspirate the MNC containing-interphase of the Ficoll gradient. The harvested MNC fraction is then subjected to several washing steps to remove potential remaining platelets. Finally, the cell preparations are verified by microscopy to be largely free of platelet contamination.
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Can I starve PromoCell HUVECs?
We have many customers who perform starvation with our HUVECs. Most of them use Endothelial Cell Basal Medium supplemented with FCS (0.5-1% for shorter periods; 5-20% for 24-48 hrs). The cells have to be in a good condition and the experiment should be terminated soon after starvation. Prolonged periods will induce apoptosis.
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What media should I use to grow PromoCell Normal Human Cells?
The recommended Growth Media are specified in the Manual belonging to the cells and can be also found on the product page of the cells (“Recommender products”).
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What are the precise localizations of both, juvenile and adult HDMEC?
Juvenile HDMEC (C-12210) are isolated from the dermis of foreskin. Adult HDMEC (C-12212) are isolated from different regions. The localizations include cheek, temple, breast, upper arm, and labia. Please contact our Technical Customer Support if you need HDMEC from a particular part of the body.
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What is the difference between PromoCell Medium “ready-to-use” and PromoCell Medium “Kit”?
PromoCell Cell Culture Media “ready-to-use” consist of basal medium and SupplementMix.
PromoCell Culture Media Kits consist of basal medium and SupplementPack.
Addition of the supplements (SupplementMix or SupplementPack, respectively) to the appropriate basal medium will result in identical growth media.Related Links and Documents
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