Cell Adaptation Protocol

Cell Adaptation Protocols

Introduction

There are two approaches to be considered when adapting cells to Sf-900 II SFM: 1) Direct planting of cells from medium containing serum to Sf-900 II SFM; 2) Sequential adaptation or “weaning". It is critical that cell viability be at least 90% and the growth rate be in mid-logarithmic phase prior to initiating adaption procedures.

Materials and Equipment:

Incubator capable of maintaining 28°C ± 0.5°C, and large enough to accommodate orbital shaker with dual platform.

Insect cell culture growth medium: Sf-900 II SFM and currently used complete medium.

Microscope, Hemocytometer

Electronic Cell Counter

Low speed centrifuge

Pipettes: 1 mL, 5 mL, 10 mL, 25 mL

Pasteur pipettes

250 mL sterile disposable Erlenmeyer flasks

A. Direct Adaptation

1) Cells growing in 5% to 10% FBS supplemented medium are transferred directly into prewarmed Sf-900 II SFM following parameters set in III, steps 1-10.

2) When the cell density reaches 1-3 x 10 6th

cells/mL (4-6 days post planting), subculture to a density of 3 x 10 5th cells/mL.

3) When the cells are completely adapted to serum-free culture they will reach a density in excess of 4 x 10 6th

viable cells/mL after approximately 7 days culture.

4) Stock cultures of SFM adapted cells should be subcultured twice weekly when the viable cell count

reaches 1 to 3 x 10 6th cells/mL with at least a 90% viability.

Note: If suboptimal performance is achieved using the direct adaptation method, use the sequential adaptation (weaning) method.

B. Sequential Adaptation / Weaning Procedure

1) Subculture Sf9 cells grown in conventional medium with 5-10% serum into a 50:50 ratio of Sf-900 II SFM and the original serum supplemented media.

2) Incubate according to III, steps 1-10 until viable cell count exceeds 6 x 10 5th cells/mL.

Subculture by mixing equal volumes of culture and fresh Sf-900 II SFM.

3) Continue to subdivide the culture in this manner until the calculated serum complement falls

below 0.1%, cell viability is approximately 85% and a viable count in excess of 6 x 10 5th cells/mL is achieved.

4) Subculture when the viable cell count reaches 1-2 x 10 6th cells/mL (approx. every 4-6 days post planting).

5) After several passages the viable cell count should exceed 4 x 10 6th cells/mL with a viability exceeding 85% after approximately 7 days of culture. At this stage the culture is considered to be adapted to Sf-900 II SFM