Human Hematopoietic Progenitor Cells (HPC) and Growth Media

These products are available in the US, Canada, and select European countries.

• Hematopoietic Progenitor Cell Culture System
• Protocols for Human CD34+ Progenitor Cell Cultivation
• Human Hematopoietic Stem Cell Culture System Quality Control
• Materials for Human Hematopoietic Progenitor Cell Culture

Hematopoietic progenitor cell culture system

CD34 is a glycosylated transmembrane protein that serves as a well-known marker for primitive blood- and bone marrow-derived progenitor cells, particularly for hematopoietic and endothelial progenitors. Although the biological functions of CD34 are largely unknown, recent data suggest that CD34 is involved in maintenance of the progenitor cells in a phenotypically undifferentiated state.

PromoCell^® CD34⁺ Progenitor Cells are isolated from the cord blood of healthy donors. CD34⁺ Progenitor Cells are suitable for diverse studies, as well as for the directed differentiation into more committed blood cell phenotypes and endothelial lineages. Immediately after isolation, the freshly prepared CD34⁺ Progenitor Cells are cryopreserved using proprietary serum-free freezing medium Cryo-SFM. Each cryovial contains at least 100,000 viable cells after thawing.

Cultivation Protocol for Human CD34⁺ Progenitor Cells

• Expansion of human hematopoietic progenitor cells in serum-free media

Hematopoietic Progenitor Cell Expansion Media was developed specifically for the expansion of HPC, and is available in two formulations. These media are provided in kits consisting of a bottle of Basal Medium and one vial of SupplementMix. Adding the SupplementMix to the Basal Medium constitutes the complete medium.

• Hematopoietic Progenitor Cell (HPC) Expansion Medium DXF is designed for the maximum expansion of primitive HPCs in a fully defined, xeno-free culture environment. Due to the use of exclusively synthetic, recombinant or plant-sourced materials, this medium is defined and free of all animal-derived components as well as substances of human origin, except for Human Serum Albumin.
• Hematopoietic Progenitor Medium (Ready-to-use) has been developed for the expansion of hematopoietic progenitors by means of differentiation into more committed lineages of blood cells. Hence, this progenitor medium is suitable for CFU-based assays like long-term culture initiating cell (LTC-IC) assays. This medium is optimized for primary human cells but can also be used for primary granulosa cells (GC) from bovine follicles, as well as primary mouse mononuclear cells.

Note: To achieve optimal performance, the complete Hematopoietic Progenitor Cell (HPC) Expansion Medium DXF must be supplemented by the user with additional growth factors. Cytokine Mix E is a ready-to-use mixture of growth factors optionally available for the HPC Expansion Medium DXF. The animal component-free, defined formula contains recombinant human TPO (thrombopoietin), SCF (stem cell factor), flt-3 ligand and IL-3. The optimized formulation allows vigorous expansion of hematopoietic progenitor cells, while maintaining their primitive phenotype and prohibiting differentiation.

Hematopoietic Progenitor Media Supplementation Details

The Hematopoietic Progenitor Medium contains all of the growth factors and supplements necessary for healthy expansion of progenitor cells. The HPC Expansion Medium DXF must be supplemented by the user with additional growth factors. Available supplements include the optionally available Cytokine Mix E (Product No. C-39890, 1 mL or C-39891, 5 mL) containing recombinant human TPO, SCF, flt-3 ligand, and IL-3. However, the user may customize the medium by supplementation with other growth factors. Hematopoietic Progenitor Media do not contain antibiotics or antimycotics and are formulated for use in an incubator with an atmosphere of 5% CO₂.

Preparation of the Supplemented Medium for Use

• Thaw the SupplementMix at 15-25 °C.
• Aseptically mix the supplement solutions by carefully pipetting up and down.
• Transfer the entire contents of each supplement to the Basal Medium.
• Close the bottle and swirl gently until a homogeneous mixture is achieved.

Important Note: For the Hematopoietic Progenitor Cell (HPC) Expansion Medium DXF, also thaw the Cytokine Mix E at 15-25 °C. Aseptically mix the solutions by carefully pipetting up and down. Transfer the entire contents to the corresponding amount of complete Medium. Close the bottle and swirl gently until a homogeneous mixture is achieved. One milliliter of Cytokine Mix E is sufficient for the supplementation of 100 mL HPC Expansion Medium DXF. Alternatively, supplement the HPC Expansion Medium DXF with growth factors of your choice.

Hematopoietic Cell Media and Supplement Storage and Stability

• Upon arrival, immediately store the Basal Medium at 4-8 °C in the dark; do not freeze the Basal Medium. Store the SupplementMix at -20 °C.
• If stored as directed, the products are stable until the stated expiration date.
• Once supplements are added to the Basal Medium, the shelf life of the complete medium is six weeks at 4-8 °C. Medium supplemented with Cytokine Mix E should be used within 2 weeks after addition of the cytokines. Do not freeze the complete medium.
• For use, prewarm only a single-use aliquot of the complete medium and keep the remaining medium refrigerated at 4-8 °C.
• After delivery, cryopreserved cells should be seeded immediately, or else stored in liquid nitrogen.
• Proliferating cells must be processed immediately.

Protocols for Human CD34⁺ Progenitor Cell Cultivation

Protocol for Cryopreserved Hematopoietic Cells

Important Notes:

• Upon arrival, store the cryopreserved cells in liquid nitrogen, or seed them immediately. Storage at -80 °C is not sufficient for cell preservation and causes irreversible cell damage.
• Handle cells using aseptic techniques and a laminar flow biosafety cabinet.
  

1. Prepare the medium
   Transfer the required volume of PromoCell Medium according to the recommended seeding density (see table below) in sterile cell culture vessels. Use at least 9 mL of medium per vial of cells. For equilibration, place the vessels in an incubator (37 °C, 5% CO₂) for 30 minutes.
   
   
2. Thaw the cells
   Remove the cryovial from the liquid nitrogen container and immediately place it on dry ice — even for brief transportation. Under a laminar flow bench, briefly twist the cap a quarter turn to relieve pressure, then re-tighten. Immerse the vial into a water or thermal bath (37 °C) just up to the screw cap for 2 minutes. Ensure that no water enters the thread of the screw cap.
   
   
3. Disinfect the vial and seed the cells
   Thoroughly rinse the cryovial with 70% ethanol under a laminar flow bench. Then, aspirate the excess ethanol from the thread area of the screw cap. Open the vial and transfer the cells to a cell culture vessel containing the prewarmed medium from step 1.
   
   
4. Incubate the cells
   Place the vessel in an incubator (37 °C, 5% CO₂) for cell attachment. Replace the medium after 4-8 hours according to the subcultivation protocol (see below).

Hematopoietic Progenitor Cell Subcultivation Protocol

1. Harvest the cells
   Harvest the cell suspension and determine the cell number. Carefully aspirate the cell suspension and transfer it to a centrifugation tube. Spin down the cells for 3 minutes at 240 x g.
   
   
2. Resuspend and reseed cells
   Discard the supernatant, add 1 mL of the appropriate PromoCell® Cell Growth Medium, and resuspend the cells by gently pipetting up and down. Seed the cells according to the recommended seeding density in new cell culture vessels containing fresh Cell Growth Medium prewarmed to 37 °C. Place the vessels in an incubator (37 °C, 5% CO₂).

Human hematopoietic stem cell culture system quality control

Human CD34⁺ Progenitor Cells Quality Control

Rigid quality control tests are performed for each lot of Human CD34⁺ Progenitor Cells.

• Routinely characterized by flow cytometry analyzing the viability, cell size, and the presence of significant markers such as CD34.
• Tested for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma).
• A detailed certificate of analysis (CoA) for each lot is available for download.

Human CD34+ Progenitor Cell Specifications

Product	Recommended Culture Media	Recommended Differentiation Media	Plating Density
Human CD34+ Progenitor Cells from Cord Blood (hCD34+-CB), single donor	Product No. C-28021 Product No. C-39891	Product No. C-28020*	5,000 cells per cm²

* Applicable for CFU-assays for hematopoietic progenitor cells (LTC-IC assays).

Hematopoietic Progenitor Media Quality Control

All lots of Hematopoietic Progenitor Media are subjected to rigorous quality control tests using primary human mononuclear cells as well as purified hematopoietic progenitor cells from cord blood. Each lot of HPC Expansion Medium DXF is tested for the ability to support the expansion of hematopoietic progenitor cells with a primitive phenotype. Each lot of Hematopoietic Progenitor Medium is tested for the capacity to induce directed differentiation into hematopoietic lineages using purified CD34⁺ progenitor cells from cord blood. Approved in-house lots of media are used as a reference. In addition, all lots of media have been tested for the absence of microbial contaminants (fungi, bacteria, mycoplasma).

Intended Use for Hematopoietic Progenitor Cells and Media System

These products are for in vitro use only and not for diagnostic or therapeutic procedures. For safety precautions please see appropriate MSDS.

Materials for Hematopoietic Progenitor Cell Culture