スフェロイドにおける哺乳動物細胞の大規模拡張のための連続供給することによって栄養規制

The overall goal of this process is to culture large number of cells under controlled nutrient conditions. Using conventional equipment found in a cell culture laboratory. The associated algorithms allow the control and manipulation of nutrients within experimental ranges.

This method can help answer key questions for large scale cell culture applications. Such as human cellular therapies. The main advantage of this technique is that is facilities culture of cells in large scale with uniform distribution of nutrients and gases.

This allows higher cell densities than static or bulk fed cell cultures, increases yield, and reduces cell loss. This can be done with equipment commonly found in most cell culture laboratories. Visual demonstration of this method may be helpful to new investigators as these techniques used can be difficult to master, due to the large number of steps that must be preformed in a specific order under sterile conditions.

Collect the five primary components needed for the feeding system. A medium reservoir, peristaltic pump, stirred bioreactor, waste reservoir, and custom designed tubing sampling set. Establish the medium reservoir using a one liter glass bottle.

The waste reservoir using a two liter glass bottle. The stirred bioreactor using a 250mL volume glass reactor. Use a digital peristaltic pump, or similar pump with a eight channel pump head to control medium exchange.

Assemble the reservoir and modified bioreactor lids from hard and autoclavable plastic. With stainless steel pipe pass-through ports to provide ventilation through sterile filters, and allow for medium transfer between reservoirs and bioreactors. Attach an outflow tube fabricated from porous glass aeration tubes to the modified stirred suspension bioreactor lid.

Assemble autoclavable perfusion tubing sets from polyvinylidene fluoride tubing connectors, and durable peristaltic pump tubing. The length of the section is dependent on the distance between the different components. Assemble the feed line using two tubing diameters.

Use L/S 14 for the primary tubing that will span the distance from the bioreactor to the medium reservoir. Insert a sort length of L/S 13 tubing in the middle of the tubing length for the pump section, using the appropriate PVDF adapters. Use standard PVDF hose-barbed tubing adapters to join two pieces of L/S 14 tubing on either side of the shorter pump section tubing.

Assemble the section tubing section for waste removal using larger diameter L/S 16 tubing, and insert a short section of L/S 14 tubing in the middle for the pumping section. This is done in the same way as the feed line assembly using PVDF hose-barbed adapters. Assemble the final component of the tubing set by constructing the set from three short L/S 14 tubing lengths.

Connected together with a T-type HVDF connector, and two small hose clamps on two of the tubing lengths. Connect the sample assembly to the stainless steel sample connector attached on the lid of the bioreactor. After sterilization, a sterile gas filter will be attached for gas venting to one of the clamped lengths.

The other is used for connection to a sterile sampling syringe. Autoclave the sampling assembly while it is connected to the bioreactor, prior to beginning the experiment as described in the text protocol. Use this assembly to collect sterile samples from the bioreactor as needed, without disturbing the continuous feed process.

Attach sterile vents to the appropriate locations on the lids of the spinner flask, the medium vessel, waste vessels, and sample ports. Culture and harvest the cells as described in the text protocol. Allow the cells to incubate at room temperature in trypsin for two to three minutes in a biosafety cabinet, and then collect them into a conical tube.

A small cell sample is then collected and stained with trypan blue. Count the cell density using a microscope and standard hemocytometer with trypan blue staining as described in the text protocol. Add the desired number of total cells to sterile stirred suspension bioreactors for each condition.

Add the medium to the stirred suspension bioreactor using a pipette to reach the desired total culture volume. Move the stirred suspension bioreactor with cells to a stir plate inside a cell culture incubator. Culture cells in the bioreactors without feeding for three days at 37 degrees Celsius, with 5%carbon dioxide, 100%relative humidity, and a stir rate of 70 rpm to allow spheroids to form.

After spheroid formation remove the stirred suspension bioreactor from the incubator. Assemble the components for the continuous feeding system in a biological safety cabinet. First fill a fresh medium reservoir with the desired culture medium and then connect to one side of the feed line.

Also ensure that the fresh medium reservoir is properly vented with a sterile filter. A sterile filter should be replaced on the bioreactor inlet port to filter the medium before it enters the bioreactor. Connect the waste line to the medium waste reservoir and ensure that the waste reservoir is properly vented with a sterile filter.

Put the stirred suspension bioreactor onto the stir plate inside of the incubator using the same culture parameters as for spheroid formation. Put the fresh medium reservoir inside a nearby mini-refrigerator. Ensure that the feed lines are able to exit the refrigerator without preventing the doors from closing.

It is also critical that the feed lines are not pinched closed by the doors of either the refrigerator or the incubator. The waste medium reservoir can be put on the bench top outside the incubator in a convenient location. Pass the remaining tubing ends into the incubator and connect the feed line to the filter on the inlet port of the bioreactor.

Then connect the waste line to the outflow tube port. Next place the pump section of the feed and waste lines into the pump head, ensuring the proper orientation so that the feed lines will pump medium from the fresh medium reservoir into the stirred suspension bioreactor, and the waste lines will pump medium from the bioreactor, and to the waste medium reservoir. Calculate the feed rate using the adjusted feed rate equation as described in the text protocol.

Use the most recent sample with cell count information, along with the growth rate prediction and the medium glucose measurements to estimate the feed rate needed to maintain the desired glucose concentration in the culture. Set the pump speed based on the calculated feed rate. Repeat the feed rate calculation and adjust the pump speed for each sampling point.

Medium glucose measurements are shown from Beta-TC6 insulinoma spheroid cultures using static stirred suspension bioreactors, and continuously fed stirred suspension bioreactor culture methods. Feeding with standard high glucose medium. The continuous feeding is able to eliminate the glucose fluctuations, but the average glucose levels in the medium change dramatically during the culture period, and are far above the physiological range.

The adjusted feeding is able to eliminate the fluctuations, as well as maintain the glucose concentrations near physiological levels for the duration of the culture period. Here cell growth reported as fold change in cell number during the 21 day culture period is compared for stirred suspension bioreactors with regular medium changes, constant feed rate stirred suspension bioreactor cultures, and adjusted feed rate stirred suspension bioreactor cultures. Following this procedure other methods like varying conditions cam be preformed in order to answer additional questions, like nutrient requirements for new cell lines, or specific experimental outcomes.

After it's development, this technique paved the way for researchers in the field of cell biology to explore metabolic requirements in cells from humans with cancer or other disorders. After watching this video you should have a good understanding of how to assemble a profusion cell culture system to better control culture conditions and maximize cell yields. Don't forget that working with human cells can be extremely hazardous, and precautions such as protection from blood borne pathogens should always be taken while performing this procedure.