Slide 1:

Making Skeletal Muscle Outside of The Body, presented by Dr. Lisa Larkin.

Slide 2:

This module will talk about a technology that Dr. Larkin is developing in her lab at the University of Michigan called the skeletal muscle unit. Our skeletal muscle unit is a scaffold-free tissue-engineered skeletal muscle.

Slide 3:

To make our engineered tissue, first we start out with a host animal. In this case, we're going to start with the sheep. We take biopsies from the animal to obtain the cells that we need to grow our tissues.

To make our skeletal muscle unit, we're going to take cells from the bone marrow and cells from a muscle called the semimembranosus muscle. The semimembranosus is a big muscle in the thigh. We take a small biopsy of each tissue back to the lab and we isolate the cells of interest from these tissue biopsies. In the case of the bone marrow, we are isolating mesenchymal stem cells and in the case of the muscle, we isolate muscle precursor cells called satellite cells.

We take the cells and we put them into tissue culture dishes in a microenvironment with media and different growth factors that allow these tissues to be induced or driven to a specific cell type.

In the case of the bone marrow cells, we drive them to a tendon lineage, and they become tendon cells.

In the case of the muscle, we drive muscle satellite cells or muscle precursor cells to an early-stage muscle cell called a myoblast.

Once we have these cells growing in their microenvironments, the first thing they do is something called proliferation, which is multiplying, dividing, and growing across the plate and filling out the entire plate.

After a couple of days, both with respect to the tendon cells and the muscle cells, they'll grow into something called a single layer or a monolayer of tissue on the plate -- basically fills the whole entire plate with a single layer of cells.

At this point, we put little pins in the plate and we switch the environment to one that will cause the cells to mature and allow the cells to interact on each other and pull themselves up off of the plate so the whole single monolayer comes off of the plate.

This is called delamination. So, the cells delaminate off the plate. If we didn't have the pins in the plate these monolayers of tissue would shrink down into little balls which would be useless to us.

We put these pins on the plate and we capture the monolayers in a column shape. We do this first with the tendon. We capture this three-dimensional shape of tendon on the pins and then we cut it in half, and we pin a piece on either side of the muscle monolayer. When the muscle monolayer delaminates and starts rolling up into that three-dimensional shape it rolls up around the tendons.

This creates our 3-Dimensional skeletal muscle unit, or a piece of muscle tissue that has tendons on either end and muscle in the middle.

We can then take that tissue that we've engineered, and we can use it in a patient, or in this case, a sheep that has a skeletal muscle injury.

Slide 4:

Going into a little bit more about the fabrication process -- the next couple slides are going to break the process down into a timeline.

The first timepoint shown is the cells in the microenvironments and induction to the appropriate lineage. In the case of the muscle, shown in the slide, we drive them to muscle or myoblast.

Myoblast are single cells –- shown in the bottom image. These cells start proliferating, or dividing, and fill out the entire plate.

At the next time point, we shift the media and we create a situation where the single muscle cells will fuse together and become myotubes.

In the bottom image, you can see these long cylindrical cells on the plate -- these are myotubes. We allow the myotubes to grow three or four days and after just a couple of days in our new environment, we get these really long muscle cells right here.

More advanced myotubes are shown in this image. The myotubes start actively contracting and they start pulling on each other until they pull themself right up off the plate. They delaminate that whole monolayer.

To capture the monolayer, we place pins in the plate. The image shown here is an actual construct.

We use the pins in the plate to add a little tiny piece of tendon tissue on either end of the muscle monolayer. The muscle rolls up around the tendon tissue and becomes a skeletal muscle unit.

In the module, I show two movies:

The movie on the left is of a muscle monolayer that is starting to roll. If you look very closely, you can see the myotubes contracting and pulling on each other and there's quite a few of them pulling on the outer edge of the monolayer. The monolayer starts rolling up and delaminating off the plate.

In the second movie, you will see a fully formed three-dimensional construct. We've removed one of the pins on the plate so that one end is free, and you can see the engineered muscle unit is spontaneously contracting in the plate.