Primer: Disposable Bag Bioreactors for Cell-Based Meat


Introduction

The success of cell-based meat will rely on the cheap and efficient production of large volumes of muscle cells. However, stainless steel bioreactors (SSBs) conventionally used in the pharmaceutical industry have high up-front capital costs, and demand expensive cleaning and sterilization in between runs. Single use, disposable bag bioreactors (DBBs) may offer an avenue to lower these costs at smaller scales.


Disposable Bag Bioreactors

Similar to traditional SSBs, DBBs have an internal compartment with an environment conducive to cell growth. While traditional SSBs need to be cleaned and sterilized after each run, DBBs have a bag that can be discarded between runs. The pre-sterilized bags are constructed with FDA-approved polymers including polyethylene, polystyrene, polytetrafluoroethylene (Teflon) and polypropylene. Components for culture maintenance and monitoring are built into the bags themselves. For example, the bags contain spargers for gas transfer and ports for sample collection, and are often equipped with sensors for measuring critical parameters like pH and dissolved oxygen.

General Electric's WAVE bioreactor utilizes rocking motion to stir the culture inside the disposable bag.

Allegro's Stirred Tank Disposable bag bioreactor uses an impeller to stir the culture inside the disposable bag.

In SSBs, the culture is generally mixed via a large propeller at the bottom of the tank. This is important to ensure that nutrients, cells, and heat are equally distributed throughout the entire tank. DBBs can achieve this by stirring via an agitator, or rocker.


Rocking Disposable Bioreactors

DBBs like GE’s WAVE and Applikon’s Appliflex mix the culture by rocking the bag back and forth like a see-saw. The rocking motion promotes gas and heat transfer and ensures nutrients are transferred to the cells and toxins away from the cells.


An advantage of using a rocking motion to mix the culture is that it is far gentler on the cells than an impeller. Impellers cause large shear forces which can disrupt sensitive cells. Additionally, rocking systems are usually cheaper than stirred tank reactors as the capital investment for a tank is not required. Furthermore, the bags themselves are cheaper than their stirred tank counterparts because they don’t require a disposable impeller to be built into each bag.


One of the major downsides of this reactor is that thermal gradients can form since heat is transferred via conduction from the heated slab under the bag. Similarly, the rocking motion may not achieve sufficient mass transfer which could limit cell growth, especially at larger scales.


Stirred Tank Disposable Bioreactors

In stirred tank DBBs the disposable bag is placed inside a stainless steel tank and acts as a liner. In this sense, they are more similar to traditional SSBs. The holding vessel is jacketed to facilitate heat transfer and the bag themselves have a built in impeller for mixing. The two main manufacturers of this type of reactor are Thermo-Fisher and Xcellerex.


In general, stirred tank bioreactors offer superior gas and heat transfer compared to rocking reactors. This ensures an even distribution of vital nutrients as well as adequate heat dissipation to maintain appropriate temperatures.

Motivation

DBBs offer some advantages and disadvantages over SSBs:


Advantages of DBBs

  1. DBBs don’t require Clean in Place (CIP) or Steam in Place (SIP) systems since the bags are replaced between runs [1]. This greatly reduces the cost of piping, automation, chemicals, and other utilities.

  2. DBBs have shorter product changeover times because replacing bags is faster than cleaning and sterilizing stainless steel tanks.

  3. DBBs have faster installation, qualification and personnel training times [2]. This is because fewer pieces of equipment need to be qualified for Good Manufacturing Practices (GMP) use.

Disadvantages of DBBs

  1. DBBs generally have lower mass transfer. Impeller geometry is limited inside the bag, and the impeller is limited in speed because of the shear stress on the bag’s seams. This can cause cells and nutrients to be unequally distributed throughout the culture.

  2. Disposable bag bioreactors are limited in scale because:

  • The seams of the bags cannot sustain the high gas pressure associated with large volumes.

  • Bags generally have poorer heat insulation than stainless steel, causing an unequal distribution of heat throughout the culture.

  • Disposable bag bioreactors have limited customization options in comparison to stainless steel reactors. Manufacturers can create custom stainless steel assemblies, which is not currently possible with plastic DBBs.

Relevance to Cell-Based Meat Production

The majority of case studies for DBBs are in the pharmaceutical industry. However, disposable bag bioreactors have several potential applications for cell-based meat production.


The production of muscle cells can be separated into two phases: a proliferation phase where the cells divide and multiply, and a differentiation phase where cells differentiate into skeletal muscle cells, then fuse into multinucleated myotubes.


Both phases require different culture environments. During the differentiation phase, a 3D support structure might be required so the skeletal muscle cells can form muscle tissue. DBBs like the FibraStage, which contain FibraCel Disks that cells can adhere to, might be useful for this differentiation phase.


Since cell-based meat will be in direct price competition with animal-based meat, the cost of manufacturing is far more important than for pharmaceutical production. DBBs could be useful in this regard since they offer considerable cost savings at smaller scales.


On a fixed-cost basis, DBBs have lower installation, qualification, and raw materials costs compared to traditional SSBs. On a marginal-cost basis, the costs of replacing bags is cheaper than CIP/SIP cleaning systems. Additionally, if multiple different types of muscle cells are being grown in a single bioreactor (beef, pork, etc.), then DBBs could offer faster product changeover times At smaller scales, the value produced by each run is lower, meaning that these costs savings could be more important.


At larger scales, SSBs might start to look better since they can justify their cost by increasing volume. As the scale and frequency of use increases, SSBs can justify their high cost and potentially become cheaper than DBBs. If DBBs are to compete with SSBs at the larger scale, bags with stronger seams capable of handling higher gas pressures would likely need to be developed.


Therefore, one major benefit of DBBs might be in the medium term, before cell-based meat companies have the resources to build manufacturing facilities with huge capacity. When companies are selling products in small volumes, it might be more economical to start with DBBs.


Due to their widespread use in the pharmaceutical industry, SSBs are more technologically developed than the novel DBBs. There are a limited number of DBB models and vendors in comparison to SSBs. SSBs also offer a far greater degree of customization in comparison to DBBs. With SSBs, cell-based meat manufacturers can design custom impellers, stainless steel assemblies and sampling ports to fit their unique needs. This is much harder to do for DBBs because all the components are made out of plastic. SSBs could lose this advantage over time if DBBs are further developed alongside cell-based meat.



Appendix



Footnotes

[1] Clean in Place (CIP) systems are comprised of a series of tanks and tubing that use chemicals like NaOH to clean and sterilize cell contacting surfaces. Steam in Place (SIP) augments CIP to further sterilize surfaces using hot saturated steam.


[2] Qualification is part of the current Good Manufacturing Practices (cGMP) and consists of a series of tasks that ensure the equipment is capable of consistently producing the desired result. cGMP is heavily used by both the pharmaceutical and food processing industries


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