Primary Human Fibroblasts and Growth Media

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

• Primary human fibroblast culture system
• Quality control of Primary Human Fibroblasts and Growth Media
• Primary Human Fibroblast Cells
• Fibroblast Growth Media
• Fibroblast Growth Media 2
• Fibroblast Growth Media 3
• Materials for Primary Human Fibroblast Culture

Primary human fibroblast culture system

Connective tissue is derived from the mesoderm and is critical for maintaining the structural integrity of the body. Fibroblasts, the predominant cells found in connective tissue, continuously secrete diverse components of the extracellular matrix. Fibroblasts exhibit unique morphology and functional properties that depend on their site of origin and physiological status.

Primary Human Fibroblasts from PromoCell are isolated from normal human juvenile foreskin or from adult skin from various sites, including adult lung tissue and aortic adventitial tissue. Soon after isolation, Human Fibroblasts are cryopreserved at passage 2 (P2) using proprietary, serum-free freezing medium, Cryo-SFM. Each thawed cryovial contains at least 500,000 viable cells. Human Fibroblasts are also available as proliferating cell cultures made from cryopreserved cells that have been thawed and cultured for three days.

• Cultivation Protocol for Primary Human Fibroblasts

Fibroblast Growth Media is available in three different formulations:

• Fibroblast Growth Medium is a serum-free medium developed for in vitro cultivation of fibroblasts. This medium is optimized for primary human cells, but can also be used for bovine, porcine, and rat fibroblasts, as well as fibroblast cell lines.
• Fibroblast Growth Medium 2 is a low-serum medium developed for the in vitro cultivation of adult Human Fibroblasts. The medium is optimized for primary human cells, but can also be used for bovine, porcine, and rat fibroblasts, as well as fibroblast cell lines.
• Fibroblast Growth Medium 3 was developed specifically for the in vitro cultivation of adult Human Cardiac Fibroblasts (HCF).

All Fibroblast Growth Media formulations are available in two options: Medium (ready-to-use) and Medium Kit. The Medium (ready-to-use) option includes a 500 mL bottle of Basal Medium and one vial of SupplementMix. The Medium Kit includes a 500 mL bottle of Basal Medium and the SupplementPack, which is a set of individual vials with pre-measured supplements, giving the user full control over the media formulation. Adding the SupplementMix or the SupplementPack to the Basal Medium generates the complete Fibroblast Growth Media. The Fibroblast Basal Medium (with or without phenol red), SupplementMix, and SupplementPack may also be purchased separately.

Recommended Media Formulations for Specific Primary Fibroblast
Cell Types

Fibroblast Growth Medium	Fibroblast Growth Medium 2	Fibroblast Growth Medium 3
Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin	Normal Human Dermal Fibroblasts (NHDF), adult donor Human Pulmonary Fibroblasts (HPF) Human Aortic Adventitial Fibroblasts (HAoAF) Human Uterine Fibroblasts (HUF)	Human Cardiac Fibroblasts (HCF)

Human Fibroblast Growth Media Supplementation Details

The Fibroblast Growth Media includes all the components necessary for the optimally growing human fibroblasts (for details see table below). Fibroblast Growth Media does not contain antibiotics or antimycotics and is formulated for use in an incubator with 5% atmospheric CO₂.

Final Fibroblast Growth Media supplement concentrations
(after addition to the medium)

Component	Fibroblast Growth Medium	Fibroblast Growth Medium 2	Fibroblast Growth Medium 3
Fetal Calf Serum	—	0.02 mL / mL	0.1 mL / mL
Basic Fibroblast Growth Factor (recombinant human)	1 ng / mL	1 ng / mL	1 ng / mL
Insulin (recombinant human)	5 μg / mL	5 μg / mL	5 μg / mL

Preparing Supplemented Media for Human Fibroblasts

• Thaw the Fibroblast Growth Media SupplementMix or SupplementPack at 15 – 25 °C.
• Aseptically mix the supplement solutions by gently pipetting up and down.
• Transfer the entire contents of each supplement to the Fibroblast Basal Medium.
• Close the bottle and swirl gently to achieve a homogeneous mixture.

Storage and Stability of Primary Human Fibroblasts and Growth Media

• Upon arrival, immediately store the Fibroblast Basal Medium in the dark at 4 – 8 °C; do not freeze. Store the Fibroblast Growth Medium SupplementMix or SupplementPack at -20 °C.
• If stored properly, the products are stable until the expiration date stated on the label.
• Once supplements are added to the Fibroblast Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8 °C. Do not freeze the complete Fibroblast Growth Media.
• To use, prewarm only the required aliquot of the complete Fibroblast Growth Medium and keep the remaining medium refrigerated at 4 – 8 °C.
• Upon arrival, store cryopreserved cells in liquid nitrogen, or seed directly.
• Proliferating cells must be processed immediately.

Quality Control for Primary Human Fibroblasts and Growth Media

Human Fibroblasts Quality Control

The following rigid quality control tests are performed on each lot of Human Fibroblasts:

• Cell morphology, adherence rate, and cell viability testing
• Flow cytometry analyses of the fibroblast-specific antigen CD90
• Growth performance testing through multiple passages up to 10 or 15 population doublings (PD) under culture conditions without antibiotics and antimycotics (see table below).
• Testing 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 Fibroblast Cell Specifications

Product	Recommended Culture Medium*	Plating Density	Passage after Thawing	Marker	Population Doublings
Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin	C-23010	3,500 - 7,000 cells per cm²	P2	CD90+	> 15
Normal Human Dermal Fibroblasts (NHDF), adult donor	C-23020	3,500 - 7,000 cells per cm²	P2	CD90+	> 15
Human Pulmonary Fibroblasts (HPF)	C-23020	3,500 - 7,000 cells per cm²	P2	CD90+	> 15
Human Aortic Adventitial Fibroblasts (HAoAF)	C-23020	3,500 - 7,000 cells per cm²	P2	CD90+	> 10
Human Cardiac Fibroblasts (HCF)	C-23025	3,500 - 7,000 cells per cm²	P2	CD90+ Smooth muscle α-actin- Slow muscle myosin-	> 15
Human Uterine Fibroblasts (HUF)	C-23020	3,500 - 7,000 cells per cm²	P2	CD90+	> 15

*catalog numbers in this table are for media in ready-to-use packaging

Fibroblast Growth Media Quality Control

All lots of Fibroblast Growth Media are subjected to comprehensive quality control testing. These tests check for growth promoting activity, adherence rate, and typical morphology of primary Human Fibroblasts. Approved in-house lots of media are used as a reference. Additionally, microbial testing ensures that all lots of Fibroblast Growth Media are free of contaminants (fungi, bacteria, mycoplasma).

Intended Use of Primary Human Fibroblasts and Growth Media

Primary Human Fibroblasts and Growth Media are intended for in vitro use only and not for diagnostic or therapeutic procedures. For safety information please see appropriate MSDS.

Note: PromoCell media have low or zero serum content and are not suitable for trypsin neutralization (when subculturing the cells, for example). Instead we recommend using the DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.

Materials for Primary Human Fibroblast Culture