A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Summary

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.  Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Abstract

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

Introduction

There is a long history of expanding rodent neural stem cells in culture as either a monolayer^1-3 or aggregate neurospheres^4-7. In addition, human neural progenitor cells (hNPCs) isolated from various regions of the developing central nervous system^8-17 have been expanded in vitro. These cells are bi-potent, capable of differentiating into both astrocytes and neurons and have been a very useful tool in studying neural development^18,19 and disease mechanism^20,21. hNPCs have also been transplanted into many different animal models of central nervous system disease with varying levels of integration, surviva....

Protocol

1. Ethical Statement and Safety

1. This procedure involves the use of cell culture products derived from humans or animals. All derived tissues must be approved before use by the appropriate Institutional Review Board(s) and/or the Institutional Animal Care and Use Committee(s).
2. All bio-hazardous waste must be disposed of according to safety regulations decided upon by the respective institution. Know and follow all of the apposite safety and disposal guidelines throughout this procedure.

2. Preparation of Equipment, Supplies, Reagents, and Observations

1. Preparation
   1. Obtain a glass Petri dish,....

Results

Figure 5. Representative data. A) Projected cell numbers of hNPCs frozen at p19, then thawed and expanded as an adherent monolayer using enzymatic dissociation compared to neurospheres passaged vi.......

Discussion

Figure 6. Chopping Schematic. Expanding spheroid stem/progenitor cells in culture using the mechanical chopping method.

Critical Steps

An overview of the chopping expansion.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Beaker, 50 ml	Fisherbrand	FB-100-50	multiple manufacturers/suppliers
Bio-Safety Cabinet, class II	Baker	SG-603A	4 ft. or 6 ft. model.  6 ft. model recommended; multiple manufacturers/suppliers
Blades, Double-edge Prep	Personna	74-0002	multiple manufacturers/suppliers.  CAUTION: Sharp
Cell Freezing Media	Sigma-Aldrich	C6295-50ML	DMSO, serum-free
Centrifuge, swing-bucket with 15 ml inserts	Eppendorf	5810 R	multiple manufacturers/suppliers
Conical Tubes, 15 ml	Fisherbrand	S50712	multiple manufacturers/suppliers
Conical Tubes, 50 ml	BD Falcon	352074	multiple manufacturers/suppliers
Controlled Rate Freezer	Planer	Kryo 750	multiple manufacturers/suppliers
Cryovials, 2 ml	Corning	430488	multiple manufacturers/suppliers
Culture Flask, Vented, T12.5	BD Falcon	353107	multiple manufacturers/suppliers
Culture Flask, Vented, T25	BD Falcon	353081	multiple manufacturers/suppliers
Culture Flask, Vented, T175	BD Falcon	353045	multiple manufacturers/suppliers
Culture Flask, Vented, T75	BD Falcon	353110	multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 1 L	Millipore	SCGPU11RE	multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 150 ml	Millipore	SCGVU01RE	multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 500 ml	Millipore	SCGPU05RE	multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 50 ml	Millipore	SCGP00525	multiple manufacturers/suppliers
Filter Paper, 8.5 cm circles	Whatman/GE	1001-085
Forceps, Standard Pattern - Serrated/Curved/18 cm	Fine Science Tools	11001-18
Freezing Chamber, Isopropyl Alcohol	Nalgene	5100-0001	"Mr. Frosty"
Incubator, 37 °C/5% CO2	Forma	370 series	multiple manufacturers/suppliers
Hemacytometer, Phase	Hausser Scientific	1475	multiple manufacturers/suppliers
McIlwain Tissue Chopper	Lafayette Instruments	TC752-PD	Petri dish modification required.   CAUTION:  Moving, sharp blade.
Micropipettor, 1-10 μl	Gilson	F144562	multiple manufacturers/suppliers
Micropipettor, 100-1,000 μl (starter kit)	Gilson	F167700	multiple manufacturers/suppliers
Micropipettor, 2-20 μl (starter kit)	Gilson	F167700	multiple manufacturers/suppliers
Micropipettor, 20-200 μl (starter kit)	Gilson	F167700	multiple manufacturers/suppliers
Nutdriver, Autoclavable, 5/16"	Steritool	10302
Pasteur Pipets, cotton-plugged	Fisherbrand	13-678-8B	multiple manufacturers/suppliers
Petri Dish, Glass, Autoclavable	Corning	3160-100
Pipet Aid	Drummond	4-000-101	multiple manufacturers/suppliers
Shim disc	McMaster-Carr	VARIABLE	multiple manufacturers/suppliers
Sterile barrier pipet tips, 10 μl	AvantGuard	AV10R-H	multiple manufacturers/suppliers
Sterile barrier pipet tips, 1,000 μl	AvantGuard	AV1000	multiple manufacturers/suppliers
Sterile barrier pipet tips, 20 μl	AvantGuard	AV20-H	multiple manufacturers/suppliers
Sterile barrier pipet tips, 200 μl	AvantGuard	AV200-H	multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 10 ml	Fisherbrand	13-676-10J	multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 2 ml	Fisherbrand	13-675-3C	multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 25 ml	Fisherbrand	13-676-10K	multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 5 ml	Fisherbrand	13-676-10H	multiple manufacturers/suppliers
Sterilization Pouches, 19 x 33 cm	Crosstex	SCL	multiple manufacturers/suppliers
Strainer, 40 µm	BD Falcon	352340
Tissue Culture Dishes, 60 mm	BD Falcon	351007
Tube Racks, Interlocking Four-Way	Fisherbrand	03-448-17
Water Bath	Fisherbrand	S52602Q	multiple manufacturers/suppliers
Neural Progenitor Cell-Specific Processing Reagents
Neural Stem Cell Expansion Medium (Stemline)	Sigma-Aldrich	S3194-500ML	Important to use the Stemline brand
Recombinant Human Epidermal Growth Factor (EGF)	Millipore	GF316	multiple manufacturers/suppliers
Recombinant Human Leukemia Inhibitory Factor (LIF)	Millipore	LIF1010	multiple manufacturers/suppliers
Trypan Blue (0.4%)	Sigma-Aldrich	T8154-100ML	multiple manufacturers/suppliers
TrypLE Select (1x)	Life Technologies	12563-011