Module 4: Process Monitoring

This is the fourth module in a four part series titled “Zero to One on the basic science behind cultivated meat”. You can click on these links to access: Course Overview, Module 1 (Cell Culture Basics), Module 2 (Cell Sources), Module 3 (Culture Medium).

By the end of this module, you should understand:

  • Two phases of cultivated meat production: Proliferation and Differentiation/Maturation;
  • Scaling up cell mass production to produce a prototype — including discussion on 2D vs. 3D (suspension) culture systems;
  • Monitoring cell viability and cellular phenotype.

The general process of producing cultivated meat requires two stages: 1) proliferation and 2) differentiation/maturation. Many of the cultivated meat companies are currently most focused on the first stage (proliferation), because this is crucial to ensuring that they can produce enough cell mass in order to produce meat products at scale. So far, somewhat less attention has been paid to the second stage.

The excerpt below from Elliot Swartz’s blog on the science of cultivated meat is helpful to introduce these concepts and also explain a few more considerations around cell proliferation:

Cell Proliferation and Immortalization

In general, the process of cultivated meat production following cell line selection can be broken up into two phases: proliferation and differentiation. In the proliferation phase, stem cells divide repeatedly to generate a large number of cells until they are transferred to a new environment and triggered to differentiate into a mature cell type via changes in scaffolding (discussed in Series III), medium composition (discussed in Series IV), or both.

One hurdle in obtaining a large number of cells is that the number of times a cell can divide is inherently limited based on the Hayflick Limit. The Hayflick Limit imposes a limitation on cell divisions due to degradation of end-capping chromosomal telomeres following each cell division. Once a certain number of cell divisions occurs (typically around 30–50 in vitro for human cells), the cells enter a state of senescence and stop dividing. Thus, the number of potential cells acquired from a single starting batch is biologically limited. Some cells, however, can bypass the Hayflick Limit and achieve cell immortality. Pluripotent stem cells achieve immortality in part by epigenetic changes (Hochedlinger and Jaenisch 2015) and up-regulation of the enzyme telomerase (Y. Huang et al. 2014), which prevents telomere degradation. This property makes pluripotent stem cells especially useful in initial scaling, although genetic drift during proliferation may also lead to cell senescence or apoptosis.

While some adult stem cells can retain some telomerase expression (Hiyama and Hiyama 2007), it is insufficient to acquire immortality.

Cell proliferation was described in module 1 (the link is copied here in case you need to refresh), but is essentially the process of a cell dividing in two to create a direct copy of itself. The cell type should stay exactly the same during this process, so a stem cell will divide to create two new and identical stem cells. Because stem cells (including satellite cells, which are stem cell-like) have better capacity for proliferation compared to differentiated cells, they are typically what is scaled up during the proliferation phase. Immortalized cells (which can include immortalized satellite or stem cells) may also be used, which gives the cells theoretically unlimited ability to proliferate. The only stem cells that inherently are ‘immortal’ (or essentially immortal) are pluripotent stem cells, such as embryonic stem cells and iPSCs. However, these cell types can be very difficult to handle and maintain in culture, because they are sensitive to their environmental conditions and are prone to spontaneously differentiating, which means they lose their stemness.

The following chart is helpful to understand the proliferative capacity of different cell types that you may want to work with. This is copied from Elliot Swartz’s blog:

Cell differentiation and maturation can occur in the second stage. These are often described as the same thing, but are in fact somewhat different processes.

Cell differentiation was introduced in module 2, but the link is copied here for your reference. Essentially, cell differentiation is the process of a cell becoming specialized. This usually refers to a stem cell turning into a specific cell type, such as a MSC or myosatellite cell differentiating into a myoblast, which is a muscle precursor cell. The process of differentiating cells depends on the cell type, but for differentiating into muscle cells, the typical procedure is based on changing the culture medium and growth factors. Specifically, the amount of growth factors present in the culture medium is decreased. The reason for this is to pull the cells out of the proliferative state, which pivots their cellular machinery towards differentiation. The sections on stem cells and satellite cells and myogenesis from Tom Ben-Arye’s review paper on Tissue engineering for clean meat production (Frontiers in Sustainable Food Systems), along with the graphic from Figure 1, go over this concept in some detail, but don’t get too hung up on the specifics here. The take home message is that changing the growth factors in the culture medium is the main way to induce differentiation.

Cell maturation is a slightly different concept, describing the process of myoblasts (muscle precursor cells) changing into myocytes, fusing together into myotubes, and then maturing into muscle fibers. This process is demonstrated in the image below, which was taken from an article on Muscle stem cell activation and proliferation (Frontiers in Cell and Developmental Biology). There’s no need to read this article in detail, but you should be aware that MuSC means muscle stem cell, which is the same as a myosatellite cell.

The process of maturation into muscle fibers is more neglected by the cultivated meat companies, and is practically very difficult to achieve. Instead, most researchers consider that fusion into myotubes is sufficient evidence of muscle formation. There is also some debate as to whether it will be necessary to mature cells all the way into muscle fibers in order to recreate the experience of eating meat. As products begin to become available, they will vary greatly in their composition (purely cell-based versus plant ingredient/animal cell hybrids), cellular maturity, and types of cells used.

Maturation can be achieved similarly to differentiation (by altering the presence of growth factors), but often there is also a need for physical cues such as mechanical tension and cellular alignment. These can be achieved to some degree using scaffolding and fabrication techniques, but this still remains challenging. This is one area that Mosa Meat is actively addressing, as briefly described in this New Harvest blog post on Mark Post’s cultured beef:

“One challenge in producing the burger was how to help the muscle fibers mature. Muscle fibers mature much better with contraction. Researchers in Mark’s lab found that seeding muscle cells around a cylinder of gel allowed the cells to create a fiber in the shape of a ring, which then could contract on itself.”

Optional: If you’d like to learn more about Mosa Meat’s approach to muscle fiber maturation, you can read analysis of their apparatus patent on Robert Yaman’s blog (Cultured Abundance).

Study questions:

1. If you start with 100,000 myosatellite cells acquired from an initial tissue biopsy, and then proliferate them for 30 population doublings at the rate of 1 doubling per day, how many cells will you end up with after 30 days?

2. If one cell weighs ~25 picograms, how much cell mass do you end up with, in kg? How much cell mass did you start with, in micrograms?

3. If one T225 culture flask (a 2D cell culture flask with 225 cm2 surface area) can hold up to 22 million cells, how many of these flasks would you use to produce the # of cells calculated in question #1?

4. What is the difference between differentiation and maturation?

5. What are the main ways to trigger differentiation and maturation?

6. In the context of recreating meat, why would you want to mature cells into muscle fibers, instead of stopping at myocytes or myotubes?

Recall the image of a bench full of stacked flasks in the New Harvest blog post on Mark Post’s cultured beef. While this can work for generating initial proof of concept at lab scale, it is still very resource and labor-intensive, and can’t possibly translate into large-scale production.

A great resource on the challenge of scale is this GFI webinar with Prof. Mark Post (Youtube). The early part of the talk goes over the general process of producing cultivated meat (and is a good summary of proliferation and differentiation), but you can skip minutes 10–34 which focus on culturing fat cells, and then watch the rest of the presentation (minutes 34–49), which discuss considerations around processing and scale-up.

Producing cells at scale requires transitioning from 2D cell culture (cells cultured on flat plastic surfaces within flasks) to 3D cell culture, where cells can be grown dispersed throughout the culture medium volume. Read the sections on Bioreactors, Anchorage-dependence and cell adaptation, and Microcarriers in Elliot Swartz’s ‘A bit of Science’ blog, which explain how this transition can be accomplished.

The chart below was copied from Elliot Swartz’s blog, and serves as a good visual summary of these concepts:

Two of the preferred 3D culture strategies are culturing adherent cells either in aggregates/spheroids or on microcarriers. These strategies enable much greater cell:culture medium volumetric ratios compared to 2D culture, making them much more efficient for scaling up cell production. Aggregate culture (culturing small clusters of cells) can be used for adherent cells and is the preferred method for culturing pluripotent stem cells, which has been optimized for human pluripotent stem cells (hPSCs) (Stem Cell Research).

  • Note: there’s no need to read this article, but just read the abstract for context.

Microcarriers are useful for other adherent cell types such as myosatellite cells and MSCs. Within the New Harvest blog post on Mark Post’s cultured beef, there is a description of Mosa Meat’s investigation into using microcarriers to scale-up the proliferation phase. The below image shows some cells (in blue) adhered to microcarrier beads (red):

A third 3D culture strategy (described in the Anchorage Dependence and Cell Adaptation section in Elliot Swartz’s blog) is to adapt adherent cells to become non-adherent, so that they can be grown as single cells in suspension.

Study questions:

7. What are the most commonly used bioreactor types for animal cell culture at scale? What are their benefits?

8. In order to generate a proof of concept at laboratory scale, what are the pros and cons of working with 2D versus 3D cell culture systems?

9. What are some of the pros and cons of aggregate culture vs. microcarriers vs. single cell suspension, for 3D cell culture?

One of the most important cell characteristics to monitor over time is the cell viability. This simply refers to assessing whether your cells are alive or not! Using a light microscope can give you a quick idea of how your cells are doing, because living cells will be spread out and attached to the culture dish (the ‘right’ cell shape to expect depends on your cell type), whereas dead and dying cells are floating, beginning to detach, or exhibiting a small rounded shape. Also recall from module 1 we discussed cell counting using Trypan blue, which can also be used to count live versus dead cells. This method is very useful to keep track of how your cells are doing during the process of feeding and passaging your cells (cell culture maintenance).

Later on, and especially once you are working with cells that might be adhered to a scaffold, or cultured in aggregates or on microcarriers, other Cell viability assays (Thermo Fisher Scientific) become useful. A commonly used assay kit is the LIVE/DEAD imaging kit. This kit is easy to use because it simply involves adding two dye reagents to your live cell sample, one of which will make dead cells (specifically the nuclei) fluoresce red, and the other makes live cells fluoresce green. The cell samples are then imaged using any standard microscope that has fluorescent imaging capabilities. Below is an example of what this looks like in practice (Image copied from LIVE/DEAD Cell Imaging Kit (Thermo Fisher Scientific)):

To quantify % cell viability, you can either count manually from a set of images, or use image processing techniques to count the # of live versus dead cells in your culture.

A quick note: Most of the cell viability and phenotype assays are sacrificial — meaning that the cells die in the process. However, this is not a problem because you only need to take a very small representative sample of your cell or tissue cultures to do these assays.

Beyond cell viability, you’ll also need to assess the Cellular phenotype. This term is defined here (Scitable), and refers to the “observable physical properties of an organism” (or cell, in this case). Cell phenotype is really important if you are working with stem cells, because you need to ensure that your stem cells remain stem cells during the proliferation phase. There are many ways to assess cell ‘stemness’, but a common method is using flow cytometry (Wikipedia) (only read the intro section) to characterize specific stem cell markers. It’s not necessary to explore this in great detail just now, but it’s something to consider depending on which cell types you are interested in using. In a practical sense, a quick clue that your cells are losing their stemness is that their proliferation capacity decreases, and you won’t see cell numbers increasing as quickly as they may have in earlier passages.

Because the purpose of proliferating cells is ultimately to have them differentiate and mature into muscle cells, it’s really important to assess whether they actually do become muscle cells! The main way to do this assessment is using fluorescence imaging techniques (Teledyne Photometrics). Cell samples are labelled with fluorescent dyes, antibodies or proteins, which can then be imaged using a fluorescence microscope. Immunofluorescence (ONI) uses fluorescently labelled antibodies to identify specific cell markers, and is a very useful and versatile technique to image just about anything in your cells that you could want to look for!

This course section on Cell Imaging — Immunofluorescence (Cellular Agriculture Course, Tufts University) has lecture slides that explain the use of immunofluorescence (also called immunocytochemistry) to image cells and tissues. Slide 6 shows the differentiation and maturation stages of muscle cells, and the text at the bottom lists cell markers that are specific to that cell type. If you look at the myotubes and mature myofibers, MyHC = myosin heavy chain, and MCK = muscle creatine kinase. These are proteins that are only expressed in fairly mature muscle cells. While there are a lot of different markers for muscle cells (R&D Systems) that you could assess, it’s most common to look for just a few key markers, such as: actin, myoD, and myosin heavy chain.

As described in the course slides, another useful way to assess muscle differentiation is by measuring the fusion index. This is measuring the # of nuclei contained within a myotube divided by the total # of nuclei within an image. Ultimately you want most of your cells to form myotubes and not exist as single cells, so the higher this number is, the more muscle-like your tissue is. For this assessment, you need to use DAPI (Wikipedia), which is a very simple fluorescent dye that binds to DNA, thereby useful for imaging cell nuclei.

Study questions:

10. What are a few different ways to assess cell viability?

11. How does immunofluorescence enable detection of almost any molecule or marker of interest in your cells?

12. What combination of techniques would you use to assess whether you have created differentiated muscle cells?

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