Method and system for suspension culture

Abstract

The present invention relates to cell culture in bioreactors, such as flexible cellbag bioreactors. More closely the invention relates to a method and system for determining the cell density in a bioreactor culture and for controlling the perfusion rate of a suspension culture of cells in a bioreactor, comprising measuring the oxygen uptake of primary mononuclear cells in a non-static bioreactor.

Claims

1. A method for regulating perfusion rate in a suspension culture of cells comprising primary mononuclear cells in a non-static bioreactor, comprising: keeping the non-static bioreactor at a constant rocking rate of 1-25 rpm; registering the dissolved oxygen concentration in the bioreactor; determining the cell number of said primary mononuclear cells by a pre-determined correlation of the registered dissolved oxygen concentration with the cell number; and automatically controlling the perfusion rate in response to the registered dissolved oxygen concentration, wherein the pre-determined correlation is an inverse linear relationship, and wherein no sampling of the contents of the bioreactor is performed to determine cell number.

2. The method according to claim 1, wherein the dissolved oxygen concentration is measured by a sensor integrated in the bioreactor.

3. The method according to claim 1, wherein the bioreactor is a flexible cell bag.

4. The method according to claim 1, wherein the cells are T-cells.

5. The method according to claim 1, wherein the rocking rate is 10-15 rpm and the bioreactor is a flexible cell bag.

6. The method according to claim 1, wherein the rocking rate of the bioreactor is 10-15 rpm.

7. The method according to claim 2, wherein the sensor is an optical sensor.

8. The method according to claim 1, wherein the dissolved oxygen uptake is greater than 10%.

9. The method according to claim 1, wherein the pre-determined correlation of the registered dissolved oxygen concentration (DO) with the cell number is defined by linear relationships:
DO=−1.58E-06*cell density+90.01 at 15 rpm and 6° angle; or
DO=−2.88E-06*cell density+89.81 at 10 rpm and 6° angle.

10. The method according to claim 1, wherein controlling the perfusion rate in response to the registered dissolved oxygen concentration comprises adjusting pump rates without operator input.

11. The method according to claim 10, wherein the pump rates control an amount of media perfused in in the non-static bioreactor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows data of an inverse linear relationship between dissolved oxygen (DO) concentration and concentration of primary human T cells in a 1 L culture on a Xuri™ bioreactor set at an angle of 6° and a rocking rate of 15 rpm of the bioreactor platform.

(2) FIG. 2 shows the relationship between DO and cell concentration when the Xuri™ bioreactor is set at a rock rate of 10 rpm and at a 6° angle.

(3) FIG. 3 is a schematic view of a bioreactor system according to the invention and shows the main features of the invention.

(4) FIG. 4 is a schematic view of a cellbag used in the bioreactor system of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

(5) The invention will now be described more closely in association with the accompanying drawings and some non-limiting Examples.

(6) The invention is a method to predict the density of primary lymphocytes grown in a bioreactor in situ. The invention uses a linear relationship between dissolved oxygen concentration and cell concentration to predict cell densities in the bioreactor culture. The invention allows perfusion rates to be set based on dissolved oxygen concentrations. A program which would allow perfusion rates to be adjusted based on dissolved oxygen readings can be written into the software that runs any selected bioreactor.

(7) The linear relationship between DO/OUR and cell concentration has never been shown for primary mononuclear cells before. The relationship was surprisingly tight and it is because of this tightness that it may be incorporated into the software control of a bioreactor, such as a WAVE or Xuri™ bioreactor. This means that cell number can be predicted and perfusion rates can be changed without having to take a sample from the cellbags.

(8) A preferred example of the bioreactor system of the invention is shown in FIG. 3 wherein (1) is a computer terminal, (2) a cellbag bioreactor control unit, (3) a gas outlet line, (4) a DO optical cable, (5) a Xuri™ Cell Expansion System, (6) a rocking platform, (7) a cellbag bioreactor, (8) a gas inlet port, (9) an optical probe for measuring DO, (10) a media waste line, (11) a media feed line, (12) and pump unit, (13) a media feed reservoir and (14) a media waste reservoir. The pump unit, the Xuri™ Cell Expansion System and cellbag Bioreactor Control Unit are connected to the computer terminal. Software in the computer terminal controls the rate that the pump unit operates which controls the rate of media exchange.

(9) FIG. 4 shows a cellbag for use in the bioreactor system of FIG. 3 comprising of (15) the fastening rods for attachment to the rocking platform, (16) a feed line, (17) a waste line, (18) a gas exhaust tube, (19) a gas inlet tube, (20) a perfusion filter, (21) an optical probe to measure dissolved oxygen, (22) a harvest port and (23) a sampling port. The perfusion filter is internal to the cellbag. The DO probe is embedded in the bottom of the cellbag and is in direct contact with the culture environment.

EXAMPLE 1

Defining the Relationship Between Dissolved Oxygen Levels and Cell Concentration

(10) Method: Peripheral Blood Mononuclear cells were cultured in static culture with anti-CD3/28 activation beads and 20 ng/ml of IL-2 for 5 days. During static culture the cell concentration was maintained at 5×10E05/ml though the addition of media. At day 5 of culture the cells were transferred to a 2 L cellbag for culture on the Xuri W25 bioreactor for a further 5-9 days. Cells were maintained at 5×10E05/ml through the addition of media until the final volume of 1 L had been reached, after which the cell concentration was allowed to increase as the cells continued to proliferate. The rocking platform was set at an angle of 6° and a rock rate of 15 rpm and kept constant throughout the course of the culture. Media perfusion was enabled once the cell concentration had reached 2×10E06/ml. The cellbags contained optical sensors for dissolved oxygen (DO) and measurement of DO levels were recorded every 24 hrs simultaneously with a cell count being taken. These data were compiled from 7 independent experiments, each using a different donor.

(11) A plot of DO verses cell concentration was drawn and a linear trendline determined (FIG. 1). The formula of the trendline in this example was
DO=−1.58E-06*Cell density+90.01

(12) Using this formula the cell density of a given culture could be predicted from the DO readings at any point during the culture period provided the rocking rate was maintained at 15 rpm and the rocking angle was maintained at 6°.

(13) The same data was generated for cultures where the rocking platform was set at 10 rpm and 6° (FIG. 2). The data was compiled from two separate experiments using 2 donors. A plot of DO versus cell concentration was drawn and a trendline with formula generated. The formula of the trendline in this example was
DO=−2.88E-06*Cell density+89.81

(14) These data show that the slope of the line is dependent on the rocking rate of the bioreactor. Therefor to use these formulas to predict cell density the rocking conditions must be kept constant throughout the culture period.

EXAMPLE 2

Predicting Perfusion Rates Based on Dissolved Oxygen Readings

(15) The ability to use DO to predict cell density can be further exploited to include an automated method of setting perfusion rates in T cell cultures. Perfusion rates are dictated by cell density, as the higher the number of cells, the higher the metabolic load and the greater the rate of media exchange that is required.

(16) The standard perfusion rates used in the above experiments were as follows

(17) TABLE-US-00001 Cell concentration Perfusion rate  2 × 10.sup.6/ml 500 ml/day 10 × 10.sup.6/ml 750 ml/day 15 × 10.sup.6/ml 1000 ml/day 

(18) Based on the trendlines of DO vs cell concentrations, the perfusion rates can be set using DO readings instead of cell concentration

(19) TABLE-US-00002 DO reading at 15 rpm Do reading at 10 rpm Perfusion rate >88 >84 none 75-88 61-84 500 ml/day 67-75 47-61 750 ml/day <67 <47 1000 ml/day 

(20) An automated set-up can be configured (FIG. 3) whereby the DO readings, which are collected continuously throughout the culture via the DO optical sensor embedded in the Cellbag bioreactor (FIG. 4) are recorded by the software and the software then adjusts the pump rates that control the amount of media to be perfused in the Cellbag bioreactor. This would eliminate the need for manual changing of the perfusion rates by the operator during the culture period. At the beginning of the bioreactor culture the operator would be required to instruct the software which perfusion rate to use based on the DO readings, as exemplified in the above table, but thereafter no further input from the operator would be required.