Technical library

PromoCell Human Chondrocytes (HCH) can be expanded in standard monolayer culture using PromoCell Chondrocyte Growth Medium (C‑27101).
This medium is an optimized formulation supplemented with 10 % FBS and is suitable for efficient cell expansion.

De‑differentiation during monolayer culture
De‑differentiation of chondrocytes is a well‑known and unavoidable phenomenon during in vitro monolayer culture and typically occurs after approximately 2 weeks.
Despite this limitation, monolayer culture is necessary to expand chondrocytes to sufficient cell numbers.

Re‑differentiation using 3‑D culture systems
Once an adequate cell number has been obtained, the monolayer system can be replaced by a more complex 3‑D culture approach, such as:

• Culture in alginate beads or gels
• Use of degradable polymer scaffolds
• 3‑D spheroid culture

Under appropriate 3‑D conditions, re‑differentiation is induced, and chondrocytes resume the production of cartilage‑specific extracellular matrix (ECM) components.

PromoCell position
PromoCell does not offer a proprietary 3‑D culture system for chondrocytes.
However, 3‑D spheroid culture combined with Alcian Blue staining is used internally by PromoCell to characterize chondrocytes during quality control.
This procedure is very similar to the protocol used for chondrogenic differentiation of MSCs.

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.

Most primary cells detach within 5-10 min at 37°C. Inactivation isn't required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.

PromoCell Human White Preadipocytes (HWP) are isolated from either subcutaneous or visceral fat tissue, which differ in location, physiological role, and disease relevance.

Subcutaneous fat
Location: Directly beneath the skin
Physiological role: Acts as the primary and physiological energy storage depot
Disease association: Generally not directly linked to obesity‑related metabolic diseases
Use case: Suitable for studies on normal adipocyte biology, energy storage, and baseline adipogenesis

Visceral fat
Location: In the abdominal cavity, surrounding internal organs (e.g. kidney, heart, bladder)
Physiological role: Expands when subcutaneous storage capacity is exceeded or adipogenesis is impaired
Disease association: Strongly linked to hypertension, type 2 diabetes, and cardiovascular disease
Use case: Preferred for research on metabolic disorders, insulin resistance, inflammation, and cardiometabolic risk
 
Functional differences
Adipocytes derived from subcutaneous and visceral fat differ in several functional aspects, including:

• Response to insulin and other hormones
• Lipolytic activity
• Endocrine and metabolic behavior

The choice between the two cell types therefore depends on the study objective.

Number of cells per cryovial
PromoCell guarantees ≥ 500,000 viable primary human cells after thawing. To ensure this, more than 500,000 cells are filled per cryovial before cryopreservation, since a fraction of cells does not survive the freeze-thaw process.
 
Viable cell number and Certificate of Analysis (CoA)
During quality control, PromoCell thaws representative vials per lot, determines viability, and calculates the number of viable cells recovered after thawing.
Both the viable cell number and the viability (%) are listed on the lot‑specific Certificate of Analysis (CoA).
Only the expected number of viable cells is reported; the total number of cells (viable + dead) is not specified.
Example:
If the CoA states 600,000 viable cells with 80% viability, the vial originally contained ~750,000 total cells, of which 600,000 were viable after thawing under PromoCell QC conditions.
No viability calculation by the user is required when the recommended thawing protocol is followed.
 
Calculating the optimal plating density
Use the number of viable cells stated on the CoA. Apply the recommended plating density for the specific cell type (e.g. 5,000-10,000 cells/cm²).
Example:
600,000 viable cells can be plated:
- into one T75 flask (~8,000 cells/cm²), or
- into one T75 + one T25 flask (~6,000 cells/cm²)

Upon arrival, PromoCell melanocytes can either be thawed immediately and seeded into a tissue culture vessel or stored in liquid nitrogen until needed.

Re‑freezing after subculture
It is technically possible to re‑freeze normal human cells, including melanocytes. However, PromoCell does not recommend repeated freezing, as each freeze-thaw cycle reduces the cells’ proliferation potential.

Recommendation
If freezing after subculture is required:

• Use PromoCell Cryo‑SFM Plus (C‑29920) as the freezing medium.
• Freeze the cells at an early passage, as this helps preserve viability and growth potential.

Baculovirus expression systems are commonly used in combination with insect cell lines (e.g. Sf‑9 or Sf‑21) to produce recombinant proteins, such as cytokines or growth factors.
PromoCell does not use recombinant proteins produced in insect cells for its media.
This applies to 

• Specialized Media for Primary Human Cells
• Blood and Stem Cell Media
• Cancer Cell Media
• Cryo‑SFM Plus Freezing Medium

None of these products contain components produced using a baculovirus/insect cell expression system.
 

PromoCell cell pellets (C-14**) can be stored indefinitely at -20°C and are stable for up to 1 month at 4°C and up to one week at room temperature. Please note: The samples do not freeze at -20°C.

Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.

When human mesenchymal stem cells (hMSC) are differentiated using PromoCell Mesenchymal Stem Cell Neurogenic Differentiation Medium (C‑28015):

• Approximately 90–100% of the cells adopt a neuronal‑like morphology.
• About 60–80% of the differentiated cells are positive for Nissl bodies, as confirmed by Nissl staining.

These results indicate a high differentiation efficiency at the morphological level, with a substantial proportion of cells also showing neuronal cytological features.