Technical Library

Recent Entries in Technical Library

• Are your cells isolated under GMP?

  Our cells are not manufactured according to GMP guidelines and are intended for in vitro use only.

  Our EXCiPACT™ GMP certification only applies to the processes related to the media.

  Related Links and Documents

  • EXCiPACT™ GMP Certification
• Can PromoCell M1 macrophages be cultured after thawing in MEM alpha containing 10% FBS and GM-CSF?

  We have never tested the cultivation of our assay-ready macrophages in MEM alpha + FBS. We cannot predict whether it will work and therefore strongly recommend the use of our M1 Macrophage Generation Medium XF and fibronectin-coated vessels.

  Related Links and Documents

  • Human monocyte-derived M1 Macrophages (GM-CSF)
  • M1-Macrophage Generation Medium XF
• Can you please provide me a protocol for the expansion of CD34+ Progenitor Cells in PromoCell Expansion Medium XF?

  Short protocol:

  • Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells
  • Spin down for 10 min at 240xg, aspirate the supernatant, resuspend the pellet at 20,000 cells/ml HPC Expansion Medium XF
  • Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂
  • Then double the media volume by adding fresh complete medium, e.g., 4 ml  suspension culture + 4 ml fresh medium (= 8 ml)
  • Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days
    Example partial medium change: For a culture volume of 8 ml, spin down the cells, aspirate and discard 4 ml of the supernatant, resuspend the cells and add 12 ml of fresh complete medium (= 16 ml).

  In combination with the Cytokine Mix E, the HPC Expansion Medium XF typically promotes a 300-1,000 fold expansion of the total cell population. After 2 weeks of expansion about 20-30% of the population express CD34⁺, indicating a 50-200 fold expansion of CD34⁺ progenitor cells.

  Related Links and Documents

  • App Note: Expansion of primitive Hematopoietic Progenitor Cells
  • Hematopoietic Stem Cell Culture
• We use Macrophage Generation Media from Promocell to get differentiated macrophages from fresh PBMC. Would it be okay if the attachment step with the Monocyte Attachment Medium is done overnight instead of 1.5 h?

  We strongly advise against overnight incubation.

  The Monocyte Attachment Medium does not contain cytokines/survival factors for the monocytes. If the cells remain in the Monocyte Attachment Medium for longer than 2 hours, they will go into apoptosis and die. The lymphocytes will survive longer. Some of them will attach after such a long time and then cannot be washed off.

  Therefore, overnight attachment is absolutely inappropriate. However, if time is short, monocyte attachment can be shortened to 1 hour. To do this, it is best to add Monocyte Attachment Medium to the culture vessels in advance and equilibrate it in the incubator. Remove the vessels from the incubator only briefly to add the appropriate amount of concentrated PBMC suspension. Then, even after 1 hour, most of the monocytes are attached.

  Related Links and Documents

  • Monocyte Attachment Medium
• Can I use a serum-free medium as negative control in the chondrogenic MSC differentiation? I would like to replace the recommended DMEM+10% FCS.

  You could probably use our MSC Chondrogenic Differentiation Medium without inducers (C-28014). It is also serum-free and the same as C-28012, just without chondrogenic inducers.

  Related Links and Documents

  • App Note: Chondrogenic Differentiation and Analysis of MSC
  • Mesenchymal Stem Cell Chondrogenic Differentiation Media
• Is it possible to thaw and re-freeze the Cytokine Mix M1 (C-39894) and M2 (C-39895)?

  The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.

  Related Links and Documents

  • M1-Macrophage Generation Medium XF
  • M2-Macrophage Generation Medium XF
• Can I use a bead bath to thaw PromoCell normal human cells?

  Based on negative feedback we have received from customers using bead baths, we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells.

  If you don’t have a “normal” water bath but only a bead bath in your lab, thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand, and not in a floater, as described in the thawing protocol.

• Do you find that the domes spread a lot on the Nunclon Sphera plates? Do they remain attached for the entire 3-4 week culture period?

  To avoid the spreading of the matrix and to have nice drop-like domes, it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help.

  From our testing, we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered, but it was able to move on the bottom and the dome became misshaped overtime. However, the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user, we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• How do you set up high throughput systems with organoids? Only in 96 well?

  It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g., Greiner Bio-One # 650185).
  A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• Why use ROCKi for organoid formation?

  ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic.

  This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:

  • https://pubmed.ncbi.nlm.nih.gov/31871466/
  • https://pubmed.ncbi.nlm.nih.gov/35169685/

  For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells