Collagen A

General description
This product is identical to Biochrom GmbH part number L 7220. It is a 0.1 % Collagen solution in HCl, type I, 1 mg/ml. Collagen A is used to coat culture flasks. Growth and differentiation of cells in vitro are significantly influenced by the substrate used (glassor plastic). If culture flasks are coated with attachment factors such as Fibronectin, Collagen, Gelatine or Polylysine, better cell growth is often achieved. Thus, for example, the survival time of hepatocytes on Collagen can be up to one month.Furthermore, due to attachment factors the tendency of many cells towards de-differentiation is prevented.
Preparation Note
1. Coating culture flasks with monomeric Collagen A
The Collagen molecules remain monomeric in solution at a pH below 3.8 and lower ion concentration, there is no fibril formation.

We recommend two different coating systems, depending on when you need the culture flasks: either for immediate use after production or after a longer storage period.

To use the culture flask immediately:
Mix 1 ml Collagen A (cat. no. L7220) with an equal volume of ultra-pure water or with 1x PBS, adjusted to a pH of 3.0 – 3.5. The PBS solution (cat. no. L1815) is adjusted to a pH of 3.0 – 3.5 as follows: Add 1 ml 1 N HCl to 100 ml PBS solution.
Add 2 ml of the diluted Collagen solution per 10 cm2 area of culture flask and incubate for 2 – 3 hours or overnight at room temperature (≤+25°C) (alternatively for 30 min at +35 – +37°C). Remove solution and wash with ultra-pure water or with 1x PBS (pH approx. 7.3). Use the culture flask immediately.
(1 ml Collagen A/10 cm = 100 μg Collagen/cm)

To store culture flasks for a longer period:
Mix 1 ml Collagen A with an equal volume of ultra-pure water.
Add 2 ml of the diluted Collagen solution per 10 cm2 area of culture flask or 20+D1 μl per well of a microtitre plate.
Incubate overnight at room temperature (≤ +25°C) in the air stream of a Laminar-Airflow system and let the coating solution dry to form a collagen film. The culture flasks can be stored sterile packed until use (cool, dry storage). Before use, rinse briefly with culture medium.

2. Coating culture flasks with fibrillar Collagen A
Collagen fibril formation is carried out at a pH above 4.0, in particular at physiological pH 7.0 – 8.0, at physiological ion concentration and warming to a maximum +37°C.

We recommend two different coating systems for fibrillar Collagen A, depending on when you need the culture flasks: either for immediate use after production or after a longer storage period.

To produce the neutralised Collagen solution cool all the solutions in an ice-water bath at approx. +2 – +8°C and mix.

To use the culture flask immediately:
Mix 2 ml Collagen A with 0.2 ml PBS solution (10x).
Adjust the pH to pH 7.3 ± 0.2 by adding approx. 10 μl 1 N NaOH.
Add 2 ml of solution per 10 cm2 area of culture flask and incubate for at least 3 hours or overnight at room temperature (≤ +25°C) (alternatively for at least 60 min at +35 – +37°C).
Remove solution and wash with 1x PBS (pH approx. 7.3) or with nutrient solution. Use the culture flask immediately.

To store culture flasks for a longer period:
Mix 1 ml Collagen A with an equal volume of 0.3 M NaCl solution and with 1 N NaOH solution, adjust to a pH of 7.0 – 8.0.
Add 2 ml of the diluted Collagen solution per 10 cm2 area of culture flask or 20 μl per well of a microtitre plate.
Incubate overnight at room temperature (≤ +25°C) in the air stream of a Laminar-Airflow system and let the coating solution dry. The culture flasks can be stored sterile packed until use (cool, dry storage). Before use, rinse briefly with culture medium.
Other Notes
Specifications:

Cell Attachment: Pass
pH: 1.8-2.4
Osmolality: 0.01-0.05 Osm/kg H20
Collagen Content: 0.09-0.15%
Hydroxyproline: 0.012-0.019%
Color: Colorless
Appearance: Clear