Primary Human Osteoblasts and Osteoblast Growth/Differentiation Media

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

• Osteoblast Cell Culture System
• Quality Control for Primary Human Osteoblasts and Osteoblast Growth Media
• Materials for Primary Osteoblast Culture

Human Osteoblast Cell Culture System

Osteoblasts are a highly specialized cells that originate from the mesenchyme. Human osteoblasts play an important role in bone formation and remodeling. In vivo, they produce the osteoid, an extracellular matrix-rich in collagen type I. During osteogenesis, the natural maturation process into osteocytes, they embed in the bone matrix and stop proliferating.

PromoCell^® Human Osteoblasts (HOB) are produced from knee and hip femoral tissue. Shortly after isolation, all Human Osteoblasts 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 Osteoblasts are also available as proliferating cell cultures generated from cryopreserved cells that have been thawed and then cultured for three days.

• Cultivation Protocol for Human Osteoblasts
• In Vitro Differentiation of Primary Human Osteoblasts into Mineralized Bone

The Osteoblast Growth Medium is optimized for culturing human osteoblasts in vitro and the Osteoblast Mineralization Medium was developed to induce robust mineralization in human osteoblast cultures with a one-step protocol, which leads to extensive extracellular calcium deposits after 21 days. These media are optimized for growing primary human cells, but can also be used for bovine, murine, and rat osteoblasts as well as osteoblast cell lines. The Osteoblast Media (ready-to-use) consists of one 500 mL bottle of Basal Medium and one vial of SupplementMix. Adding the SupplementMix to the Basal Medium constitutes the complete Osteoblast Media. The Osteoblast Basal Medium (with or without phenol red) and Osteoblast Growth Medium SupplementMix are also available for separate purchase.

Osteoblast Media Supplementation Details

The Osteoblast Growth Medium contains all the necessary components for the optimal growth of human osteoblasts. The Osteoblast Mineralization Medium contains all the components necessary for mineralization of human osteoblasts. Osteoblast Media do not contain antibiotics or antimycotics and are formulated for use in an incubator with 5 % atmospheric CO₂.

Final Osteoblast Growth Medium supplement concentrations (after addition to the medium)

Component	Final Concentration
Fetal Calf Serum	0.1 mL / mL

Preparing Supplemented Media for Primary Human Osteoblasts

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

Instructions for use of Osteoblast Mineralization Medium

• Plate 3x10⁴ HOB cells per well on a collagen I-coated 24-well tissue culture plate.
• Work in duplicate: Use HOB Growth Medium (C-27001) for one well as a negative control and Osteoblast Mineralization Medium (C-27020) for the other well.
• Incubate the cells for 17 – 21 days. Change the medium every third day, working carefully to avoid disturbing the cell monolayer.
• Fix the cells, and stain for osteogenic markers.
• Note: For detailed information, refer to protocol In Vitro Differentiation of Primary Human Osteoblasts into Mineralized Bone

Storage and Stability of Osteoblasts and Growth/Differentiation Media

• Upon arrival, immediately store the Osteoblast Basal Medium in the dark at 4 – 8 °C; do not freeze. Store the Osteoblast SupplementMix at -20 °C.
• If stored as directed, the products are stable until the expiration date stated on the label.
• Once supplements are added to the Osteoblast Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8 °C. Do not freeze the complete media.
• To use, aliquot and prewarm just the volume of the complete Osteoblast Media needed for single use, and maintain 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 Osteoblasts and Growth/Differentiation Media

Human Osteoblasts (HOB) Quality Control

The following rigid quality control tests are performed for each lot of Human Osteoblasts:

• Cell morphology, adherence rate, and cell viability testing
• Histochemical testing for alkaline phosphatase and bone mineralization
• Growth performance testing through multiple passages up to 10 population doublings (PD) under culture conditions without antibiotics and antimycotics
• 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 Osteoblast Specifications

Product	Plating Density	Passage after Thawing	Marker	Population Doublings
Human Osteoblasts (HOB)	10,000 – 20,000 cells per cm²	P2	Alkaline phosphatase+ Mineralization tested	> 10

Osteoblast Growth and Differentiation Media Quality Control

All lots of Osteoblast Growth Medium are subjected to comprehensive quality control testing. These tests check for growth-promoting activity, adherence rate, and typical cell morphology using primary human osteoblasts. Further, all lots of Osteoblast Mineralization Medium are tested for their capacity to induce mineralization in primary human osteoblasts. Approved in-house lots of Osteoblast media are used as a reference. Additionally, microbial testing ensures that all lots of Osteoblast Growth/Differentiation Media are free of microbial contaminants (fungi, bacteria, mycoplasma).

Intended Use for Human Primary Osteoblasts and Osteoblast Growth/Differentiation Media

Human Osteoblasts and Growth/Differentiation 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 our DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.