Automated Cell Culture: A Closer Look
Demo of the Formulatrix Cellmatic and Associated Liquid Handlers
Intro
I’ve made many connections with vendors since starting this blog, and one of the benefits of that is receiving high quality media that I am able to share that shows liquid handlers in action. In this post, I want to give a closer look, with videos, of what an automated sub-culturing protocol looks like, a deeper dive into the FLO i8, and a brief introduction into scheduling software. Special shout out to the team at Formulatrix for the videos they’ve allowed me to share with you today.
Cell Splitting Protocol
For those of you who have not had the pleasure of performing cell culture, cell sub-culture, also known as cell splitting, is a way to maintain cells for use in scientific research. The bigger picture is, a sub-population of cells is harvested and transferred to a new receptacle to grow. This is necessary to prevent cell overgrowth which can cause epigenetic changes in daughter cells that will cause them to behave differently adding variability to your studies. Another important note is that splitting cells causes an increase in passage number of a cell line. Think of a passage number as the age of a cell line that starts from when they are thawed and ends when they are disposed of. There is often a passage number range where the performance of a cell line is optimal. Once a cell line has reached the end of its optimal range, you toss the cell line and thaw a new vial. This starts the passage number from the beginning, so starting with a validated cell bank is important.
This protocol will focus on splitting cells that have been thawed. I’m going to list the steps in the process, share a video clip, and explain what is happening in the clip to give you some clarification. Let’s get started:
Appropriate medium for the cell line being split is transferred from the bottle into a sterile plastic trough with a lid. Trypsin will be dispensed into a separate trough.
An SBS trough for media is carted on a Rover over to a refrigerated unit
A set volume of a specific reagent is dispensed into the trough.
The lid is added to maintain sterility in the trough.
The reagent in the trough is warmed up. Medium must be warmed up before coming into contact with cells to reduce cellular stress which can cause epigenetic changes in daughter cells.
An infrared lamp is used to warm media to 32C quickly.
The Rover acts as the plate “mover” placing the trough into the media warmer and retrieving it for the next step of the process.
The Rover retrieves the plate to be processed from the incubator.
6 Rovers move about in the Cellmatic to move media storage troughs and cell plates throughout the Cellmatic.
All plates and troughs are barcoded for proper sample tracking.
Cell plates are trypsinized in the Reagent Exchanger.
The Reagent Exchanger has two dispense lines: one stationary, plumbed in line and a second one for switchable troughs. The plumbed in line will dispense PBS.
The trough containing trypsin and the cell plate are transported via Rover to the Reagent Exchanger.
The plate is tilted which allows aspiration pins to aspirate media without coming into contact with the monolayer while maximizing the amount of liquid being aspirated.
PBS is dispensed with the 8 channel bulk dispenser. Here, the tilted plate allows for gentle dispensing on the side of the plate, minimizing cell damage from mechanical force.
PBS is aspirated and trypsin is added with the second dispense line from the trough
A Rover transports the plate to the incubator so the trypsin can digest the proteins that keep the cells attached to the flask.
The Rover moves the plate to the FLO i8 to deactivate trypsin and reseed the cells into new flasks containing pre-warmed media.
Rovers move the cell plate with trypsinized cells, warmed media trough, tips, and two new empty cell plates onto the deck.
All plates and troughs are delidded with suction cups
The FLO i8 aspirates media and dispenses it into the new cell plates for seeding
The tips are chucked and new tips are grabbed; fresh media is aspirated
Cells are dislodged by dispensing media starting from the top of the plate and then down along the surface
Cells are mixed to create a uniform suspension
Cells are transferred from the mother plate to the daughter plate at a desired volume based on their doubling time
The plates are moved back and forth to create a uniform cell suspension that will settle into a uniform monolayer
Rovers move daughter plates into the incubator until the next passage
If you want a more precise cell passage, here are some alternate steps you can take:
Rover moves cell plate from incubator to on-deck imager
Before splitting cells, the on-deck imager can use brightfield imaging to measure confluence of your cell plate
The splitting protocol will trigger based on the confluence measurement, and the cells will be inoculated based on a split-ratio if the minimum confluence measurement is met.
If the cells aren’t confluent, then the Rover will move the cells into the incubator to grow and take a measurement at a later time point based on what is programmed in the scheduling software.
This is best for new cell lines that you don’t know the doubling time for. The Cellmatic collects data for you to help calculate the doubling time leading to more consistent cell splitting.
After trypsinization, cells can be counted for the most precise splits
Media is added to deactivate trypsin and cells are mixed to create a uniform suspension.
The FLO i8 takes an aliquot of cells and mixes it with trypan blue in a dilution plate at a specified ratio.
Trypan blue dilution plate can be added in another step by the FLO i8 or it can be added by hand and placed into the robot for the Rovers to move into place.
The diluted sample is transferred to the cell counting plates.
A Rover moves the cell counting plate to the imager and calculates the viable cell density(VCD) and viability which is: (viable cells / total cells) * 100.
Dead cells are measured by imaging blue cells stained by Trypan blue
Total cells = viable cells + dead cells
With the VCD measured, a set number of live cells can be transferred to a new receptacle for growth.
This is also perfect for generating cell solutions used to seed cell-based assay plates at a given density.
FLO i8 Liquid Handler and Positive Displacement Liquid Handling
The workhorse liquid handler in this instrument is the FLO i8 PD Liquid Handler. The videos show the FLO i8 with air dispensing, but that is being phased out and replaced with the FLO i8 PD, which uses positive displacement. Each Cell system will be retrofitted with the new positive displacement dispenser. I’ll talk more about positive displacement in a later post. Here are the specs:
0.2uL - 1mL dispense range
8 channels that can move in the X,Y, and Z axes
LLD (liquid level detection)
Touch probes in each channel, good for finding the side or bottom of the well and detecting unexpected collisions
Tip-based positive displacement liquid handling
Non-contact dispensing
Can dispense 0.2µL in a 384 well plate in 15 seconds
3% or lower CV when dispensing 1µL to 1000µL
5% CV when dispensing 500nL with contact dispense and 10% CV dispensing 200nL with non-contact dispense
I also wanted to highlight positive displacement. I covered some dispensing technologies in this post. Positive displacement uses a pump to dispense precise volumes of fluid regardless of their liquid class. Think of a syringe, which has a plunger that comes into contact with the liquid. For this dispenser, the tips allow for sterile positive displacement, which is crucial to any cell culture workflow.
Scheduling Software
The Cellmatic comes with its own web-based control software that has been specifically designed with cell culture in mind. I can talk about scheduling software more in-depth in a subsequent post, but think of it as a way to give a robot commands that you want carried out at a later time.
Scheduling software is crucial for walk away automation which saves scientists evening and weekend trips to the lab. This all comes down to the data the instrument is able to collect and making that data available to a user that is off site. When familiarizing yourself with a new cell line, taking measurements like cell counts and confluence will tell you a cell line’s doubling time, or the time it takes a cell line to double in population size. Being able to remote into the instrument and make a decision that the robot can carry out is a big step towards fully automating your cell culture.
Once enough data is collected, you can feel more confident in splitting cells on a set schedule without any intervention. In both cases, a scientist does not need to be onsite for the cells to be split.
Conclusion
My main goal with this post was to show a cell culture protocol done with automation. Hopefully seeing the Formulatrix Cellmatic in action gave a clearer picture of automated liquid handling technologies in the context of cell culture; additionally, I gave a deeper dive into the FLO i8 liquid handler and introduced scheduling software. The Cellmatic unlocks the potential for fully automated cell culture, so visit their website and talk to a rep if you’d like to learn more!
Also wanted to share my guest spot at The Automated Lab, complete with illustrations by one of the founders, Madeline Wolf! I’ve shared some of their content on my blog before, and I was honored to have been able to work with them. It really is a great resource! I write about cell culture and automation, very relevant to today’s post.
I know I promised an announcement, but you’ll just have to wait until my next post!