An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Summary

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Abstract

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Introduction

This article aims to provide actual and detailed handling procedures for an automated culture system for human induced pluripotent stem cells (iPSC), which we produced by collaborating with a company, and to show representative results.

Since the publication of the article in 2007, iPSC has been attracting attention all over the world¹. Due to its greatest feature of being able to differentiate into any type of somatic cell, it is expected to be applied in various fields such as regenerative medicine, elucidating the causes of intractable diseases, and developing new therapeutic drugs^2,3. In addition, using human iPSC-derived somatic cells could reduce animal experiments, which are subject to significant ethical restrictions. Although numerous homogeneous iPSCs are constantly required to research new methods with iPSCs, it is too laborious to manage them. Moreover, handling iPSC is difficult because of its high sensitivity, even to subtle cultural and environmental changes.

To solve this problem, automated culture systems are expected to perform tasks instead of humans. Some groups have developed a few automated human pluripotent stem cell culture systems for cell maintenance and differentiation and published their achievements^4,5,6. These systems equip multiarticulated robotic arm(s). Robotic arms have not only merit in that they highly mimic human arm movements but also demerit in that they require higher cost(s) for the arm(s), larger and heavier system packaging, and time-consuming education efforts by the engineers to obtain the aimed movements^7,8. In order to make it easier to introduce the apparatus to more research facilities at the points of economic, space, and human resource consumption, we have developed a novel automated culture system for the maintenance and differentiation of iPSC into various cell types⁹.

Our rationale for the new system was to adopt an X-Y-Z axis rail system instead of multiarticulated robotic arms⁹. To replace the complex hand-like functions of robotic arms, we applied a new idea to this system, which can automatically change three types of specific functional arm tips. Here, we also indicate how users can easily make task schedules with simple orders on software because of the lack of requirements for engineers' contributions throughout the process.

One of the robotic culture systems has demonstrated the making of embryoid bodies using 96-well plates as 3D cell aggregates for differentiation⁴. The system reported here cannot handle 96-well plates. One achieved the current good manufacturing practice (cGMP) grade using a cell line, although it was not a human pluripotent stem cell⁵. The automated culture system detailed here has now been developed with the specific aim of helping laboratory experiments (Figure 1). However, it has enough systems to keep clean levels equivalent to a level IV safety cabinet.

Protocol

The Ethics Committee of the Kansai Medical University approved the generation and use of the healthy volunteer-derived iPSCs named KMUR001 (approval No. 2020197). The donor, who was openly recruited, provided formal informed consent and agreed with the scientific usage of the cells.

NOTE: The current interface (the special software named "ccssHMI" running under the Windows XP operating system) is the fundamental operation screen. Under the aforementioned interface, a series of tabs are arranged, allowing users to initiate various operations.

1. Loading operations

1. Click the Loading button on the software's top screen. Click the Loading Preparation Start button.
2. Place the dish(es) or plate(s) to be entered into the apparatus in position in the apparatus.
   NOTE: Necessary information for dish identification should be written on each lid.
3. Manually close the front sliding window and push the mechanical safety-confirmation button.
4. Select the type and quantity of dish(es) or plate(s) in the software.
5. Click the Loading Preparation Completed button. Click the Loading Start button.
6. After a dish is uploaded into the system, select the information on the dish, such as with iPS cells or without iPS cells, on the software. Register notes about each dish in the software.
7. Click the Registration button at the end to complete the loading operation.

2. Unloading operation

1. Click the Unload button on the software's top screen. Select the dish(es) to be removed in the software.
2. After selecting the dish(es), click the Unloading Preparation Start button. Click the Unloading Start button.
3. After the dish(es) have been transferred from the incubator to the workbench in the system, press the Dish Removal button.
4. Manually open the front sliding window and take out the dish(es). Manually close the front sliding window and push the mechanical safety-confirmation button.

3. Supplement of consumables: pipettes, tubes, and medium

1. To replenish consumables such as pipettes, tubes, and medium, click the Consumables button on the software's top screen, then select the item to be replenished.
   1. Pipettes
      1. Click the Pipette button. Select the Replenish button.
      2. Select a rack that the user wants to replenish on the software. Click the Replenish Start button.
      3. After confirming that the lid of the pipette storage area on the workbench is opened, manually open the front sliding window and replenish the pipettes as needed.
      4. Manually close the front sliding window and push the mechanical safety-confirmation button. Click the Replenish Completed button.
      5. Click the Replenishment Setup button and enter the information for the replenishment, then click the Registration button.
      6. Click the Replenish Completed button.
   2. Tubes
      1. Click the Tube button. Select the Replenish button.
      2. Select a rack that the user wants to replenish on the software. Click the Replenish Start button.
      3. After confirming that the rack has moved to the top, click the Replenish Tube button.
      4. Manually open the front sliding window and replenish the tubes as needed.
      5. Manually close the front sliding window and push the mechanical safety-confirmation button.
      6. Click the Replenish Completed button.
      7. Click the Replenishment Setup button and enter the information for the replenishment, then click the Registration button. Click the Close button.
   3. Medium
      1. Click the Medium button. Select the Replenish button in the software.
      2. Select one rack from three that users want to replenish. Click the Replenish Start button.
      3. After confirming that the lid of the medium storage area has been opened, manually open the front sliding window and replenish the medium.
      4. Manually close the front sliding window and push the mechanical safety-confirmation button.
      5. Click the Replenish Completed button.
      6. Click the Replenish button and enter the information for the medium, including the name and the amount of the medium. Enter additional comments if necessary.
      7. Click the Registration button.
      8. Click the Close button.

4. Task selection

1. Click the Task button on the software's top screen.
2. Select the Task Setting button. Select the desired task from the task list and click the Next Step button.
3. Specify the date and time to perform the task, and then click the Registration button. Select a dish or plate to perform the task, and then click the Registration button.
4. After reconfirming the selected task, click the Registration button. Confirm that the task has been registered on the next screen.
5. If necessary, set the next task in the same way.
6. Click the Start button at the end. Then, the task will start automatically at the specified date and time.
   NOTE: Immediately after finishing every task, UV lights (located on two sides of the hood) automatically turn on, and after 5-30 min, in accordance with the auxiliary setting, they are turned off to keep the hood in aseptic condition. To stop, users can click the Start button.
7. If users want to cancel a scheduled task in advance, click the Stop button.
8. After selecting the task to be aborted, click the Edit Task button.
9. Click the Task Cancel button. Confirm that the task has been deleted on the next screen.

5. Check cell pictures

1. Every task includes microscopic observations(taking photos) before and after the task work. Observe the progress of cell culture photographically by incorporating a microscopic photography task before or after each task.
2. Select pre-set task programs, including selecting multiple specific locations of the dish or the well plate in advance for fixed-point observation to monitor the same location over time.

6. Passaging and differentiation

1. Follow the steps in sections 1-5 and set the automated cell culture system to perform passaging and differentiation. The instrument settings for passaging, cardiomyocyte differentiation, hepatocyte differentiation, neural precursor cell differentiation, and keratinocyte differentiation are shown in Table 1, Table 2, Table 3, Table 4, and Table 5, respectively.

Results

Maintenance of human-induced pluripotent stem cells
We used three hPSC lines (RIKEN-2F, 253G1, and KMUR001). We have optimized the maintenance protocol through daily manually performed experiments and further optimized the detailed programs through the seven preliminary experiments performed by the system. For example, shear stresses caused by the liquid speeds of the spit flow from different pipets handled by humans and the system are quite different; therefore, we optimized the time length of the...

Discussion

A critical step in the protocol is that if a user finds any faults, click the cancel, stop, or reset button anytime and start over from the first step. The software can avoid human mistakes, including double booking, opening doors while the system tasks are active, and a lack of replenishment. Another critical point for successful and efficient differentiation to the desired somatic cell is the proper selection of pluripotent stem cell lines because each pluripotent stem cell has an uncontrollable bias in its differentia...

Materials

Name	Company	Catalog Number	Comments
0.15% bovine serum albumin fraction V	Fuji Film Wako Chemical Inc., Miyazaki, Japan	9048-46-8
1% GlutaMAX	Thermo Fisher Scientific	35050061
10 cm plastic plates	Corning Inc., NY, United States	430167
253G1	RKEN Bioresource Research Center	HPS0002
2-mercaptoethanol	Thermo Fisher Scientific	21985023
Actinin  mouse	Abcam	ab9465
Activin A	Nacali Tesque	18585-81
Adenine	Thermo Fisher Scientific	A14906.30
Albumin  rabbit	Dako	A0001
All-trans retinoic acid	Fuji Film Wako Chemical Inc.	186-01114
Automated culture system	Panasonic
B-27 supplement	Thermo Fisher Scientific	17504044
bFGF	Fuji Film Wako Chemical Inc.	062-06661
BMP4	Thermo Fisher Scientific	PHC9531
Bovine serum albumin	Merck	810037
CHIR-99021	MCE, NJ, United States #HY-10182	252917-06-9
Defined Keratinocyte-SFM	Thermo Fisher Scientific	10744019	Human keratinocyte medium
Dexamethasone	Merck	266785
Dihexa	TRC, Ontario, Canada	13071-60-8	rac-1,2-Dihexadecylglycerol
Disposable hemocytometer	CountessTM Cell Counting Chamber Slides, Thermo Fisher Scientific	C10228
Dorsomorphin	Thermo Fisher Scientific	1219168-18-9
Dulbecco’s modified Eagle medium/F12	Fuji Film Wako Chemical Inc.	12634010
EGF	Fuji Film Wako Chemical Inc.	053-07751
Essential 8	Thermo Fisher Scientific	A1517001	Human pluripotent stem cell medium
Fetal bovine serum	Biowest, FL, United States	S140T
FGF-basic	Nacalai Tesque Inc.	19155-07
Forskolin	Thermo Fisher Scientific	J63292.MF
Glutamine	Thermo Fisher Scientific	25030081	Glutamine supplement
Goat IgG(H+L) AlexaFluo546	Thermo Scientific	A11056
HNF-4A  goat	Santacruz	6556
Hydrocortisone	Thermo Fisher Scientific	A16292.06
Hydrocortisone 21-hemisuccinate	Merck	H2882
iMatrix511 Silk	Nippi Inc., Tokyo, Japan	892 021	Cell culture matrix
Insulin-transferrin-selenium	Thermo Fisher Scientific	41400045
Keratin 1  mouse	Santacruz	376224
Keratin 10  rabbit	BioLegend	19054
KMUR001	Kansai Medical University		Patient-derived iPSCs
Knockout serum replacement	Thermo Fisher Scientific	10828010
L-ascorbic acid 2-phosphate	A8960, Merck	A8960
Leibovitz’s L-15 medium	Fuji Film Wako Chemical Inc.	128-06075
Matrigel	Corning Inc.	354277
Mouse IgG(H+L) AlexaFluo488	Thermo Scientific	A21202
N-2 supplement	Thermo Fisher Scientific	17502048
Nestin mouse	Santacruz	23927
Neurobasal medium	Thermo Fisher Scientific	21103049
Neurofilament  rabbit	Chemicon	AB1987
Neutristem	Sartrius AG, Göttingen, Germany	05-100-1A	cell culture medium
Oct 3/4  mouse	BD	611202
PBS(-)	Nacalai Tesque Inc., Kyoto, Japan	14249-24
Rabbit IgG(H+L) AlexaFluo488	Thermo Scientific	A21206
Rabbit IgG(H+L) AlexaFluo546	Thermo Scientific	A10040
Recombinant human albumin	A0237, Merck, Darmstadt, Germany	A9731
Rho kinase inhibitor, Y-27632	Sellec Inc., Tokyo, Japan	129830-38-2
RIKEN 2F	RKEN Bioresource Research Center	HPS0014	undifferentiated hiPSCs
RPMI 1640	Thermo Fisher Scientific #11875	12633020
SB431542	Thermo Fisher Scientific	301836-41-9
Sodium L-ascorbate	Merck	A4034-100G
SSEA-4  mouse	Millipore	MAB4304
StemFit AK02N	Ajinomoto, Tokyo, Japan	AK02	cell culture medium
TnT rabbit	Abcam	ab92546
TRA 1-81 mouse	Millipore	MAB4381
Triiodothyronine	Thermo Fisher Scientific	H34068.06
TripLETM express enzyme	Thermo Fisher Scientific, Waltham, MA, United States	12604013
Trypan blue solution	Nacalai Tesque, Kyoto, Japan	20577-34
Tryptose phosphate broth	Merck	T8782-500G
Wnt-C59	Bio-techne, NB, United Kingdom	5148
β  Tublin  mouse	Promega	G712A