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.
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.
There is a long history of expanding rodent neural stem cells in culture as either a monolayer1-3 or aggregate neurospheres4-7. In addition, human neural progenitor cells (hNPCs) isolated from various regions of the developing central nervous system8-17 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 development18,19 and disease mechanism20,21. hNPCs have also been transplanted into many different animal models of central nervous system disease with varying levels of integration, survival and functional effects22-24.
Traditionally, rodent or human fetal-derived NPCs are exposed to growth factors – often epidermal growth factor (EGF) and/or fibroblast growth factor-2 (FGF-2)25-28 – and both adherent29 and three-dimensional spheroid systems are typically passaged using enzymatic dissociation into a single-cell suspension30-34. The standard method to expand cells for research or clinical use is as an adherent monolayer due to easy manipulation. However, we have shown that passaging monolayer and neurosphere hNPCs with enzymatic or chemical solutions resulted in early senescence35. In addition, enzymatic dissociation may result in increased levels of differentiation and karyotypic abnormalities based on data demonstrated with embryonic stem cells36-38. Although the standard method of passaging hNPCs has produced current good manufacturing practice (cGMP) grade products that have gone into phase 1 clinical trials (Stem Cells Inc., Neuralstem Inc.), the method permitted only a few rounds of cell amplification, limiting the banking potential.
Clearly, large research experiments and future clinical trials could benefit from the ability to propagate cells in bulk and with delayed senescence to permit large-scale growth and cell banking. To address this need, we developed a novel and automated way of mechanically passaging intact neurospheres by “chopping” them into small clusters to maintain cell-to-cell contact. This method greatly increased their lifespan39 and suspension culture permits a more efficient use of incubator space compared to monolayer cultures, as seen with an alternative 3D bioreactor culture method40. The provided chopping protocol allows for the production of large-scale banks from one fetal sample greater than passage 10, an unlikely feat using standard passaging methods. While this method for passaging hNPCs is unconventional, it is growing in popularity and was recently, published with other cell types such as neural stem cells derived from human embryonic and induced pluripotent stem cells, enabling large scale expansion for various applications including in vitro disease modeling41-46. Importantly, a cGMP-grade hNPC cell bank has already been produced with the chopping method, demonstrating that the technique can be applied towards future clinical applications.
1. Ethical Statement and Safety
2. Preparation of Equipment, Supplies, Reagents, and Observations
Flask | Volume of total media | Pre-Chop | → | Post-Chop |
1 T12.5 | 5 ml | 1 T12.5 | → | 1 T25 |
1 T25 | 10 ml | 1 T25 | → | 1 T75 |
1 T75 | 20 ml | 1 T75 | → | 1 T175 |
1 T175 | 40 ml | 1 T175 | → | 2 T175s |
2 T175s | 80 ml | 2 T175s * | → | 4 T175s |
* 2 T175s is the maximum number of flasks that can be chopped at a time. Chop in sets of 2 T175s and refer to step 7. |
Table 1. hNPC expansion paradigm. Description of a typical expansion scheme for hNPCs. It is standard to expand two-fold volumetrically every 7-10 days.
3. Chopper Setup
Figure 1. McIlwain Tissue Chopper. A) Chop thickness adjustment micrometer, B) Chopper arm base and attached arm, C) Hook on plate holder for Petri dish, D) Table release knob and tray, E) Blade force control knob, F) Reset switch, G) Plate holder, H) Bolt attachment for blade, clasp and nut, I) Nut wrench included with chopper, J) Blade/clasp nut, K) Blade clasp, L) Automated chopping speed control knob, M) Manual chopping arm operating knob, N) Power switch.
4. Pre-chop Procedure
Figure 2. Sphere preparation for chopping. A) Lean the flask(s) against a tube rack or similar item to settle the spheres in the corner of the flask. B) Transfer the spheres as densely as possible from the conical tube to the Petri dish. C) Pool the spheres from the conical tube in the middle of the Petri dish. D) Remove as much supernatant as possible from the top of the pooled spheres. E) Spread the spheres out using the side of a plastic micropipettor tip. F) Gently move the spheres to one side of the pool. G) Example of spheres that have been moved to one side of the pool to facilitate media removal. H) Condensed spheres spread out on the Petri dish, ready for chopping. Click here to view larger image.
Column A | Column B | Column C | Column D | Column E | Column F | |
Pre-Chop Flask Size | → | Post-Chop Flask(s) Size | Suggested volume of MM to transfer to new flask(s) pre-chop | Suggested volume of CM to transfer to new flask(s) pre-chop | Suggested volume of spheres/media to transfer into new flask(s) post-chop | Final Volume Seeded/Flask |
T12.5 | → | T25 | 5 ml | 0 ml | 5 ml | 10 ml |
T25 | → | T75 | 10 ml | 0 ml | 10 ml | 20 ml |
T75 | → | T175 | 20 ml | 10 ml | 10 ml | 40 ml |
1 T175 | → | 2 T175s | 20 ml per flask | 15 ml per flask | 5 ml per flask | 40 ml |
2 T75s | → | 4 T175s | 20 ml per flask | 17.5 ml per flask | 2.5 ml per flask | 40 ml |
Table 2. Media transfer guide pre/post-chop. Suggested volumes to use during the chopping process.
5. Chop Procedure
6. Post-chop Procedure
7. Process Variations – Multiple Flasks
There are several differences when passaging more than two T175s. The steps below are alterations of the referenced step.
8. Cryopreservation
The following protocol is for cryogenically preserving hNPCs.
Figure 3. Fire-polishing glass Pasteur pipets. A) Hold the pipet in the top part of the flame and spin in order to evenly round the edges of the glass pipet. B) Example of a large-bore fire-polished glass pipet. C) Example of a small-bore fire-polished glass pipet.
Step | Rate (°C) | End Temperature (°C) | Hold (min sec) | Trigger |
1 | — | — | 5 min 0 sec | Chamber |
2 | – 1.3 | – 5 | — | Sample |
3 | — | — | 1 min 0 sec | Chamber |
4 | – 45 | – 58 | — | Chamber |
5 | + 10 | – 26 | — | Chamber |
6 | + 3 | – 23 | — | Chamber |
7 | – 0.8 | – 40 | — | Sample |
8 | – 10 | – 100 | — | Chamber |
9 | – 35 | – 160 | — | Chamber |
Table 3. Steps for freezing hNPCs in a controlled rate freezer. Suggested program for hNPC cryopreservation on a controlled rate freezer.
Figure 4. Sample freezing curve. Typical freezing curve for hNPCs on a controlled rate freezer.
9. Thawing Procedure
The following protocol is for thawing cryogenically preserved hNPCs.
# of vials | Total Seeding Volume (ml) | Flask |
1 | 5 | T12.5 |
2 | 10 | T25 |
3 | 15 | T75 |
4 | 20 | T75 |
5 | 25 | T75 |
6 | 30 | T175 |
7 | 35 | T175 |
8 | 40 | T175 |
8 + | — | Combination of Flasks |
Table 4. Flask sizes based on the number of cryovials thawed. Suggested volume and flask size to seed hNPCs post-thaw.
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 via the chopping method. Day 0 represents when the cells were thawed at p20. B) Representative images of spheres pre-chop, 10X. C) Representative images of spheres post-chop, 10X. D) Color swatch used to describe the extent of media conditioning in cGMP-comparable protocols. E) Immunocytochemistry of thawed p29 hNPCs that were plated for 1 day and stained for nestin (red) expression. Hoechst nuclear counter stain (blue). F) Immunocytochemistry of thawed p29 hNPCs that were plated for 7 days and stained for GFAP (Red) and β–III tubulin (Green). Click here to view larger image.
Figure 5A represents hNPCs thawed at p20 (day 0) and maintained adherent to laminin-coated tissue culture flasks or as non-adherent neurospheres passaged via the chopping method. The adherent cells were enzymatically dissociated using TrypLE Select weekly and senesced after 70 days (7-10 passages) in culture. In contrast, the neurospheres thawed at passage 20 and expanded via the chopping technique grew for greater than 40 passages before senescence. Typical hNPCs before chopping (Figure 5B) should be mostly ≥ 300 µm in diameter. Once chopped, the spheres are generally quartered into variably sized sections, mostly around 200 µm in diameter (Figure 5C). It is important to note that hNPCs expanded via the chopping method maintain the expression of hNPC markers and can produce both astrocytes and neurons post-differentiation. Late passage hNPCs were dissociated, plated onto laminin-coated coverslips for a period of one or seven days and subsequently fixed with 4% paraformaldehyde. The hNPCs express the progenitor cell marker nestin (Millipore, 1:1,000) on 1 day plated hNPCs (Figure 5E) as well as the differentiated neural markers glial fibrillary acidic protein (GFAP, 1:500) and β–III tubulin (1:2,000) on the seven day plated hNPCs (Figure 5F), markers for astrocytes and immature neurons, respectively.
Figure 6. Chopping Schematic. Expanding spheroid stem/progenitor cells in culture using the mechanical chopping method.
Critical Steps
An overview of the chopping expansion paradigm is shown in Figure 6. hNPC sphere size is one of the important criteria to observe before passaging the neurospheres. Although there is a large variance in sphere size, at least 30% of the spheres should have a diameter greater than 300 µm. If the spheres are too small to passage, simply exchange the CM with fresh MM (25%-50%) and re-assess after 1-4 days. Poor expansion rates occur when the spheres are passaged at too small of a diameter.
hNPC expansion rates are also dependent on sphere density and media conditioning due to secreted factors47. However, if the media is overly metabolized and important growth factors are diminished, the cells may acquire a karyotypically abnormal population of cells48. Therefore, replacing nutrients and growth factors must be balanced with secreted trophic factors. When seeding hNPCs post-chop or post-thaw, the number of neurospheres per cm3 of media is variable. However, the general rule is the greater the density, the faster the media will condition and the higher the expansion rate, provided the media is exchanged when needed. Use the media-conditioning color spectrum (Figure 5D), which ranges from pink when un-metabolized, to yellow when the media has been fully metabolized. The ideal media color for logarithmic growth is a reddish-orange color, #3 on the color swatch. This indicates the media contains sufficient nutrients and growth factors while maintaining an adequate concentration of secreted trophic factors. The media should not condition past #4 on the color swatch. Alternatively, when the cells are not metabolizing media quickly (#1 on the color swatch), the hNPCs will grow slower and require a chop every 10-20 days as opposed to every 7-10 days. It is important to increase the culture density to boost conditioning, which will in turn improve expansion rates.
During the chopping procedure note the following critical steps. Ensure the blade is installed parallel to the chopper arm. If the blade is not flat on the surface of the Petri dish or shim disc, many spheres may not be chopped. Note the spread of cells discussed in step 4.11. The spheres must remain in the center of the Petri dish or the blade will contact the wall before all spheres have been chopped. Finally, it is important to avoid drying the hNPCs for an extended period of time during the chopping process. After placing spheres in the dish, chop and flood them with fresh media as soon as possible.
As this technique requires several pieces of equipment, there can be risks to the sterility of the culture. Given this risk, proper sterile technique must be used throughout the procedure. For basic research level production, wiping down all equipment with 70% IPA or equivalent is sufficient along with optional ultraviolet incubation for a minimum of 15 min. Perform all steps inside of a sterile BSC. For cGMP level production, the chopper must be sterilized with ethylene oxide as suggested by the manufacturer. All other supplies can be purchased sterile or autoclaved.
Future Applications
The transition of any basic research therapy from the bench to the bedside can be a challenge. The chopping procedure was designed with this transition in mind. The procedure has already been used to produce a cGMP-compliant hNPC bank at the University of Wisconsin Waisman Center Bio-manufacturing Facility. That hNPC bank was sourced for the expansion of a pharmaceutical-grade hNPC cell lot that will be used for new pre-investigational drug studies and a phase 1 clinical trial following Federal Drug Administration approval. There are other cells types that use this procedure for expansion. An example are EZ spheres, neural stem cells generated from pluripotent stem cells41. This technique has great potential for use with other types of tissue that require constant cell-to-cell contact during expansion.
The authors have nothing to disclose.
We thank Dr. Soshana Svendsen for critical review and editing of this report. This work was contributed to by the NIH/NINDS 1U24NS078370-01 and CIRM DR2A-05320.
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 – 1000 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, 1000 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 |