Method

1 Dissociate the monolayers of differentiated hES cells in wells using EDTA solution (as described in Protocol 7, Steps 7-9); filter the cell suspension, and count the filtered single cells (Step 10).

2 Resuspend the cells at 3-5 x 105 cells/ml in hVEGF-supplemented VDM seed onto 35 mm bacteriological-grade Petri dishes using 6 ml per dish, and incubate for 12-24 h to induce cell aggregation in suspension.

Collagen assay:b

3 Collect the cell aggregates from Step 2, centrifuge for 3 min at 1500 rpm and 4 °C, and resuspend aggregates from each 35 mm dish in 1 ml of a mixture of equal volumes of 2 x alpha MEM and collagen Type I solution, precooled on ice.a

4 Dispense 250 ml of the cell suspension per well into either 24-well or 4-well dishes, and incubate for 15 min at 37 °C to allow gelling to occur.

5 Supplement the cultures with 500 ml hVEGF-supplemented VDM per well and incubate. Sprouting and network formation should appear ~72 h after seeding.

Matrigel™ assay:b

3 Thaw the undiluted Matrigel™ solution overnight at 4 °C, dispense 380 ml per well of either 24-well or 4-well dishes, and incubate at 37 °C for 30 min to allow gelling to occur.

4 Collect the cell aggregates from Step 2, centrifuge for 1 min at 800 rpm and 4 °C, and resuspend aggregates from each 35 mm dish in 2 ml hVEGF-supplemented VDM.c

5 Seed 250 ml cell suspension onto the Matrigel™ layer and incubate. Vascular sprouting and network formation should appear 24-48 h after seeding.

6 The extent of vascular network formation, and the properties of those networks, can be studied using various qualitative and quantitative approaches: microscopic measurements may be taken to monitor the kinetics of sprouting and branching, and total capillary tube length; and transmission electron microscopy and immunos-taining may be used for morphological and cellular characterization.

a It is important to perform this procedure on ice, to preserve a liquid-gel status. b For both assays, vascular initial sprouting and tube-like structures can be observed to form within 48 h of culture, using an inverted phase-contrast microscope (Figure 8A). c The reduced time and speed of centrifugation are to preserve the integrity of the aggregates.

6.1 Dynamic systems for EB production

At the present time, the aggregation of multiple hES cells is an obligatory process to initiate efficiently EB formation. Established methods for producing EBs (45, 46) include culture of hES cells in hanging drops, suspension culture in liquid medium using non-adhesive substrata, and culture in semisolid medium containing methylcellulose (MC). These systems all involve 'static' culture in Petri dishes, are laborious and time-consuming, are not controllable, and therefore are impractical for industrial scale up. And so we have introduced 'dynamic' systems for the large-scale production of EBs, which are collectively termed 'dynamic' because of the motion applied to maintain and equilibrate the internal environment. Our prototype systems are described below.

Any dynamic system for EB production must strike a balance between allowing ES cells to aggregate, which is necessary for EB formation, and causing EBs to adhere and to agglomerate into large masses, which is detrimental to their uniform growth and differentiation (49). Our initial attempts at producing differentiating EBs from mouse and human ES cells cultured in suspension in spinner flasks resulted in the formation of large clumps of cells within a few days, which is indicative of a significant tendency of both mES cells (50) and hES cells (personal observations) to aggregate under these conditions. However, the high rate of stirring that was required to avoid EB agglomeration was accompanied by massive hydrodynamic damage to the cells from turbulence and collision between the fragile, incipient EBs and the apparatus. Therefore, to circumvent these problems we employed a static system for an initial aggregation period of 4 d, followed by a period in dynamic culture in spinner flasks, to successfully achieve the bulk production of cardiomyocytes from differentiating mES cells (51).

Another, more technically advanced, dynamic approach that is highly effective for hES cells is to generate and culture EBs within rotating cell-culture systems (RCCStm; manufactured by SYNTHECON™ Inc.) originally developed by NASA for tissue culture in zero gravity (52; Figure 9A). These bioreactors provide exceptionally supportive environments for the three-dimensional organization and differentiation of human tissue (further details and information are available on the website, http:/www.synthecom.com). In the RCCSTM the operating principles are:

(a) Whole-body rotation around a horizontal axis, with cells or aggregates in permanent free-fall. The resulting flow pattern in the RCCSTM is laminar with mild fluid mixing, as the vessel rotation is slow. The settling of cell clusters, which is associated with oscillation and tumbling, generates the fluid mixing. The net outcome is a very low-shear, dynamic environment allowing efficient mass transfer, and from which turbulence and the collision of cells are absent.

(b) Oxygenation by active or passive diffusion across a membrane to the exclusion of all but dissolved gasses from the reactor chamber. Crucially, the vessel is devoid of gas bubbles and gas/fluid interfaces, which are otherwise deleterious. An added advantage of the RCCSTM is that the vessels are geometrically designed so that the membrane-area to volume-of-medium ratio is high, thus facilitating efficient gas exchange.

Figure 9 (see Plate 14) Mass production of EBs by a rotating cell-culture system. (A) Cross-sectional diagram of the rotating bioreactor (STLVtm) used for the formation of small EBs from hES cells (Biotechnology and Bioengineering copyright (2004); copyright owner as specified in that Journal). As the vessel rotates slowly around its horizontal axis, hES cells aggregate in suspension with the minimum of shearing force. Note the absence of a medium-to-air interface found in conventional bioreactors. (B-D) Haematoxilin- and eosin-stained sections of EBs generated after 1 month in rotation culture, showing a variety of cell types including: epithelial neuronal tubes (dashed arrows), blood vessels (arrowheads), connective tissues, and cyst formation (solid arrows). Note that these dynamically produced EBs are more uniform in size and shape than those obtained in static suspension culture, as demonstrated in Figure 3. Scale bars = 100mm.

Figure 9 (see Plate 14) Mass production of EBs by a rotating cell-culture system. (A) Cross-sectional diagram of the rotating bioreactor (STLVtm) used for the formation of small EBs from hES cells (Biotechnology and Bioengineering copyright (2004); copyright owner as specified in that Journal). As the vessel rotates slowly around its horizontal axis, hES cells aggregate in suspension with the minimum of shearing force. Note the absence of a medium-to-air interface found in conventional bioreactors. (B-D) Haematoxilin- and eosin-stained sections of EBs generated after 1 month in rotation culture, showing a variety of cell types including: epithelial neuronal tubes (dashed arrows), blood vessels (arrowheads), connective tissues, and cyst formation (solid arrows). Note that these dynamically produced EBs are more uniform in size and shape than those obtained in static suspension culture, as demonstrated in Figure 3. Scale bars = 100mm.

With regard to EB formation, culture, and differentiation in particular, these bioreactors offer: an optimized environment for hES cell aggregation; minimal hydrodynamic damage to incipient EBs; reduced opportunity for EB fusion and agglomeration (the speed of rotation may be increased according to EB size, to this end); and the uniform growth and differentiation of EBs in three dimensions, as they oscillate and rotate evenly (see below). We recommend the Slow Turning Lateral Vessel™ (STLVtm) for this purpose.

Human ES cells cultured within the STLV™ aggregate by 12 h, in contrast to 48 h for hanging-drop or suspension culture. The average diameter of dynamically formed EBs after 1 week of culture is about 20 mm, and from day 7 onwards the total number of EBs does not change significantly but their size increases with time, reaching 400-800 mm after 1 month of culture: this compares with 400-1500 mm for EBs in static culture. Furthermore, the use of a STLV™ has a significant impact on the overall process of EB formation from hES cells: in addition to their smaller size, the STLVTM-borne EBs exhibit evenly rounded shape and uniform size, with minimal agglomeration, and three-fold

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