Developmental Biology Applications for the Sony SH800 Cell Sorter

"There are a lot of unanswered questions about early heart development," says Peter Andersen, PhD, a research associate in Dr. Chulan Kwon’s lab in the Department of Cellular and Molecular Medicine at Johns Hopkins University. "About 30% of spontaneous abortions are due to cardiac abnormalities, and about 1% of newborn babies are born with a congenital heart defect. But we know very little about this because it occurs during development in the uterus, and for obvious reasons we cannot study that in humans."

"We've developed this 'time machine' that significantly speeds up the process of maturation into adult cells."

What's the alternative? "We turn to the mouse," says Dr. Andersen, who shares expertise with Dr. Kwon in mouse genetics and mouse biology. Years ago, they began trying to model these early developmental events, starting with embryonic or induced pluripotent stem cells (PSCs) and differentiating them into cardiac progenitor cells and then into cardiac muscle cells (cardiomyocytes, or CMs).

There they ran into a wall. "These cells cannot grow to adulthood in a dish," Dr. Andersen explains. "In vitro, they remain immature, like baby cells, embryonic cells. So when we try to model diseases in these cells, they behave very differently from adult CMs. For example, drugs that are effective on adult cells might not be effective on these baby cells."

 

A Bioincubator Breakthrough

Drs. Andersen and Kwon found an ingenious workaround.¹ If the mouse cardiac progenitor cells are injected into neonatal rat hearts, they mature normally into adult CMs. Even more of a breakthough: human cardiac progenitor cells do the same! The rat heart becomes a "bioincubator" for generating mature mouse or human CMs.² Nor do researchers have to wait long for them to mature. "What's powerful about this method is that while it takes 15 or 20 years for a human baby to reach adulthood, the rat becomes adult in about 8 to 10 weeks. And our human cells incubated there do the same. We've developed this 'time machine' that significantly speeds up the process of maturation into adult cells." The technique is shown visually in Figure 1.

In this research, the lab relies heavily on their Sony SH800 cell sorter. "We use the SH800 in many different aspects of our work," says Dr. Andersen, "I cannot think of an application where we are not using it." In the CM maturation study, the researchers had to demonstrate that the incubated cells were truly adult. They used the Sony SH800 to sort individual in vitro human and mouse CMs into plates based on GFP and RFP expression transfected into the underlying PSC lines, and compared them with in vivo adult CMs. "We performed many different functional and genomic assays on them," Dr. Andersen reports, "and showed that they were identical to the real deal." Figure 2 shows 3D imaging of a matured CM.

 

 

Currently, the lab has multiple projects that combine single-cell sorting with RNA sequencing (RNA-Seq). "This combination has been extremely powerful and high throughput," Dr. Andersen says. "We are able to sort thousands of cells into a 384-well plate, process them, and send them off to RNA-Seq." In one study, they developed a cardiac organoid method to study development of the two cell populations - the First and Second Heart Fields (FHF and SHF) - that give rise to different parts of the heart. They sorted the cells by labeling the FHF and SHF cells with GFP and RFP respectively. "We showed that we can make these organoids and mimic this process in a dish. They were almost indistinguishable from in vivo mouse cells. That was amazing to see." Figure 3 shows a cluster analysis demonstrating that adult (green) and in-vivo-matured (blue) CMs were genomically similar to each other, but very different from in-vitro-matured CMs (red).

 

Game Changer: Automation

Dr. Andersen was one of Sony's first SH800 customers back in 2013. Why did he choose to pioneer a new cell sorter? "The game changer for me was automation," he says. Other vendors' sorters required a full-time operator to spend an hour or more calibrating the machine in the morning, then monitor it constantly "because just a small change can change everything."

Sony offered a fully automated machine that would calibrate, stabilize, and monitor itself. "I was skeptical," Dr. Andersen recalls, "but when I tested it out, it was exactly right. I didn't need more lasers. What I really need is a machine that can handle itself, that I don't have to train and pay an expert to sit and watch the machine all the time. In my mind, that's why Sony has been so successful. They listened to customers and developed a product that really worked."

Dr. Andersen also emphasizes the user friendliness of Sony's software. "Imagine engineers who are designing software for biologists. They design it so they understand it, but engineers don't usually speak the same language we do, so the software was very hard to use. Sony made software that is user friendly, easy to use, and very intuitive." It was also easier to train new users. "We have a big lab with a lot of people who need the sorter. I don't have time to do all of it for them. With the Sony, I can train a person in 30 or 45 minutes. After that, they are able to start up the machine, run the sort, and shut it down again. That's amazing."

 

"Walkaway sorts are very efficient for us. With the Sony, you can put your sample on, start the sort, and literally walk away."

A second game changer for Dr. Andersen was the walkaway sorting. "Walkaway sorts are very efficient for us," he reports. "Other machines require constant monitoring by an expert. With the Sony, you can put your sample on, start the sort, and literally walk away. Twenty minutes to an hour later, depending on the number of cells, you come back to sorted cells, which you can plate as you want. It's made my life a lot easier and our day-to-day workflow far more effective."

 

Solving Special Sorting Challenges

Dr. Andersen faces several special sorting challenges in his work. "Cardiomyocytes are very big - up to 200 µm in length and 20 µm wide - rectangular, and very stiff," he explains. "We use the biggest sorting chip we can - the 130-µm chip from Sony. We have to dilute the samples; otherwise the chip can clog. But using our protocols, the sorter is gentle enough to actually sort live adult functional myocytes! It's not necessarily high throughput, but it's far faster than picking them in a dish."

Because the Kwon lab makes their Sony SH800 available to other research groups in the department, the disposable fluidic chips are also useful in managing multiple users and sample types. Each experiment is run with a fresh fluidic chip. "With non-myocytes, we typically use the 100-µm chip, which covers 95% of the cells of interest," Dr. Andersen says. "That makes it easy for us, and eliminates any cross-contamination problems between samples."

Although Dr. Andersen finds it logistically simpler to reserve the SH800 for sorting, he occasionally uses it to analyze the sorted cells. "We have a two-laser flow cytometer [cell analyzer] as well as the Sony sorter, so I tend to divide it up. But as a four-laser system, the Sony can detect up to six colors - two more than on the cell analyzer. So if we need more than four colors, we turn to the Sony."

Finally, what's the impact of having a cell sorter right in the lab? "It's huge," Dr. Andersen says. "It makes us a much more effective lab. When equipment like a cell sorter is shared in a core facility, it's very hard to get time slots on it. We work with patient cells and rarely get 24-hour notice that a patient is coming in. When they undergo surgery and the cells are available, we need to get on the sorter right away."

Of course, word gets around. "Just today I received two email requests from Hopkins researchers requesting access to our sorter. It's made me a very popular man!" he says with a laugh.

 

 

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References

1. Cho GS, Lee DI, Tampakakis E, et all. Neonatal transplantation confers maturation of PSC-derived cardiomyocytes conducive to modeling cardiomyopathy. Cell Rep. 2017; 18: 571-582. PubMed
2. Cho GS, Tampakakis E, Andersen P, Kwon C. Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells. Nat. Protoc. 2017; 12: 2097-2109. PubMed