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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

Key Words. Embryonic stem cells ? Embryoid bodies ? Scalable bioreactor ? Stirred-suspension culture ? Microcarriers ? Aggregates ? E-cadherin

Correspondence: Peter W. Zandstra, Ph.D., Institute of Biomaterials and Biomedical Engineering, Room 407, Roseburgh Building, 4 Taddle Creek Road, Toronto, Ontario, Canada M5S 3G9. Telephone: 416-978-8888; Fax: 416-978-4317; e-mail: peter.zandstra@utoronto.ca% L0 C/ x- A& Z5 G% e6 T  e5 {! H

ABSTRACT) X2 d. s+ T1 r3 U0 j7 V$ \$ _
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To facilitate the investigation and exploitation of embryonic stem cell (ESC)–derived cells in research, scale-up of cell production and optimization of culture conditions are necessary . To date, however, there have been few advances in the development of scalable culture techniques for the propagation of undifferentiated ESCs . Because the mouse is widely exploited as a model for conducting in vitro and in vivo studies to gain insight into different aspects of developmental biology and regenerative medicine that may eventually benefit humans, it is important to develop a technology for the scalable and controllable production of murine ESCs and ESC-derived cells.6 \1 v" c4 `1 M& e* T3 H
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ESCs are currently cultured as a monolayer on tissue-culture plastic and are subject to variations in the cellular microenvironment due the batch-wise passaging conditions. The rapid exhaustion of cultivation area in this system necessitates frequent user intervention and is associated with a heightened risk of contamination. Most importantly, the batch-type approach does not provide steady-state operating conditions and adequate cell physiology control, leading to variations in the cellular microenvironment  (potentially leading to uncontrolled cell-differentiation responses). Thus, new methods for the scale-up and control of ESC cultures are required. Stirred cultures are particularly appealing because of their ability to provide robust spatial and temporal control . Relevant to ESC growth and differentiation, stirred-suspension cultures have been used to control cell aggregation and 3-D tissue development . Adherent cells can also be expanded in stirred suspension using microcarriers as substrate for cell attachment .

One challenge associated with the suspension culture of ESCs and ESC-derived cells is their recognized dependence on cell–cell adhesion and aggregation for propagation . In this report, we examine the use of hydrodynamic shear to control aggregation and agglomeration. Impeller-associated shear effects have been shown to influence the aggregate size of baby hamster kidney  cells and neural stem cells . The control of aggregate size is important in the regulation of cell agglomeration  because the centers of very large aggregates (>300 μm) may experience cell death due to limitations in nutrients and oxygen delivery . It has been reported that the minimum shear needed to remove cells cultured on surfaces is 6.5 dyn per cm2 , whereas shear stress levels in the range of 15–30 dyn per cm2 are shown to cause damage to attached cells . Therefore, a culture system that can control aggregate size effectively must exert a shear stress that falls within the aforementioned ranges.. [/ R3 n, a" g; E! J# Q) H" s- d

We report the establishment of two stirred-suspension systems, one using microcarriers as a substratum for ESC growth and a second that used shear to control ESC aggregate size. Both systems could be used to maintain an expanding ESC population; the second system could also be used to generate ESC-derived cells directly upon leukemia inhibitory factor (LIF) removal.! e" I- I/ W3 }; `" b4 H  d
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MATERIALS AND METHODS8 G1 `" H0 k$ y/ x. ]
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ESC Growth on Microcarrier Surfaces! e0 p" w3 X$ x
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Cultivation area restriction is a major concern associated with the culture of ESCs on tissue-culture plastic. Therefore, we explored the feasibility of adapting existing culture techniques to address this problem. The use of microcarriers in stirred suspension was an appealing alternative because the microcarriers have a high surface area–to–volume ratio, and the available cultivation area in each culture vessel can be adjusted easily.

Twelve commercially available microcarriers were used to establish ESC cultures in Petri dish–grade six-well plates. Glass microcarriers (SoloHill Engineering, Inc.) and Cytodex 3 micro-carriers (Amersham Bioscience) were chosen for the stirred-suspension studies based on the extent of ESC growth and/or culture morphology such as cell spreading (data not shown).

Total cell numbers were determined on days of harvest based on cell counts performed using trypan blue exclusion. The net population doubling time was calculated assuming that the viable cells exhibited exponential growth kinetics between seeding and harvest with a negligible lag phase. The extent of cell growth was also assessed via cell fold expansion, which was defined as the ratio of the viable cell number at harvest per 50-ml culture volume to the viable seeding density. The total or cumulative fold expansion, determined at the end of the 15-day culture period, was simply the sum of the cell fold expansions determined on days of harvest. CCE and R1 cells could be cultured on microcarrier surfaces consistently throughout the culture period. In particular, the average net population doubling times recorded for CCE and R1 cells cultured on glass microcarriers (CCE: 13.9 ± 0.7 hours; R1: 17.0 ± 1.9 hours) were not significantly different from their respective tissue-flask controls (CCE: 14.8 ± 1.3 hours; R1: 17.2 ± 2.0 hours) (Fig. 1A). CCE cell cultures could also be established on Cytodex 3 microcarriers, although they exhibited a slower net population doubling time (17.0 ± 2.4 hours). In contrast, R1 cells could not be cultured consistently on Cytodex 3, yielding poor and variable net population doubling times. CCE cells cultured on glass microcarriers achieved cumulatively a 191.8 ± 11.3–fold expansion during the 15-day culture period (compared with the 112.9 ± 10.5–fold and 158.8 ± 27.8–fold expansions achieved by the CCE Cytodex 3 cultures and tissue-flask controls, respectively) (Fig. 1B). R1 cells cultured on glass microcarriers had similar total fold expansions as their corresponding tissue-flask controls (105.3 ± 12.4–fold versus 104.4 ± 10.6–fold, respectively), but R1 Cytodex 3 cultures expanded only approximately 30-fold during the 15-day culture period (Fig. 1B).
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Figure 1. Undifferentiated ESCs could be cultured on microcarriers for an extended period of time via serial passaging. (A): Net population doubling times for CCE (filled bars) and R1 (hollow bars) cells cultured on tissue-culture flasks, glass-coated styrene microcarriers, and Cytodex 3. Data are expressed as mean of three independent experiments ± SD. (B): Cumulative cell-fold expansions for CCE (filled bars) and R1 (hollow bars) cells cultured on tissue-culture flasks, glass-coated styrene microcarriers, and Cytodex 3. Data are expressed as mean of three independent experiments ± SD. (C): Fifteen-day average of percentage of positive SSEA-1 expression detected in viable CCE (filled bars) and R1 (hollow bars) cells harvested from tissue-flask control as well as glass and Cytodex 3 microcarrier cultures. Data are expressed as mean of at least two independent experiments ± SD. (D): Fifteen-day average of percentage of positive E-cadherin expression detected in viable CCE (filled bars) and R1 (hollow bars) cells harvested from tissue-flask control as well as glass and Cytodex 3 microcarrier cultures. Data are expressed as mean of at least two independent experiments ± SD. (E): Oct-4 expression was quantified on each day of harvest plus on days of EB analyses for tissue cells obtained from () tissue-flask control, () Cytodex 3, and () glass microcarriers. Data are expressed as mean of at least two independent experiments ± SD. (F): Representative dot plots of ESCs cultured using the three techniques and stained for Flk-1 and CD34 on day 15 of undifferentiated culture as well as day 4 and 9 of EB formation. (G): Reverse transcription–polymerase chain reaction analysis of ESCs and differentiation marker expression on day 15 ESC culture, day 4 and day 9 of EB culture. (H): Images capturing the morphology of ESCs (CCE) were obtained at x100 magnification for cultures established on (i) glass and (ii) Cytodex 3 microcarriers. The arrowheads indicate the sites of bead-bridging. Occasionally, ESCs were able to grow as even monolayers on microcarrier surfaces, as shown in the insets. Abbreviations: EB, embryoid body; ESC, embryonic stem cell.7 D) j8 ?- W% Z4 s) e

To support the above cell-growth analyses, ESC characteristics were examined by monitoring the maintenance of ESCs in their undifferentiated state during the culture period, as well as their differentiation potential using flow cytometry and RT-PCR analysis. SSEA-1, E-cadherin, and Oct-4 expression was measured every 3 days during the 15-day culture period. The cells were then used to generate EBs to analyze ESC differentiation through the detection of markers from the three germ layers: Flk-1, CD34, MHC, HNF3-? and Sox-1. Regardless of the culture method, SSEA-1 and E-cadherin expression measured using flow cytometry remained high (>80%) for both CCE and R1 throughout the culture period (Figs. 1C and 1D, respectively). Oct-4 expression was also maintained (>80%), with a subsequent downregulation upon EB formation (Fig. 1E). Flk-1 and CD34 expression on day 15 of the culture was low; Flk-1 expression was upregulated by day 4–5 of EB formation, followed by the expression of CD34 by day 8–10 of EB formation (Fig. 1F). Flk-1, HNF3-?, MHC, and Sox-1 genes were upregulated as cells differentiated, as detected by RT-PCR (Fig. 1G).8 l! t( |& x" K+ _- F7 D! I; w( M

A significant amount of bead-bridging (indicated by arrowheads in Figure 1H), caused by the collision of microcarriers and the adhesion of cells at the points of contact , was observed in both the glass and Cytodex 3 microcarrier cultures (Fig. 1H). Ideally, the surface of each microcarrier should be evenly covered with a monolayer of cells. Bead-bridging created a suboptimal culture environment, and hence the appropriate ratio of beads to cells could not be determined.
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ESC Expansion as Spontaneously Formed Aggregates in Stirred Suspension
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From the bead-bridging study we noticed that the resulting cell aggregates were not detrimental to ESC growth, indicating that these cellular masses contained live ESCs. Because the maintenance of microcarrier cultures can be laborious and time-consuming, we set out to develop a method to culture ESCs in suspension without a surface onto which cells adhered, allowing the cells to be sampled and harvested with ease.0 f" ~4 a1 r7 U2 U! g

CCE and R1 cells formed aggregates spontaneously in continuously stirred cultures. Fifteen-day cell-growth data for the stirred-suspension CCE and R1 cell cultures maintained at 60 and 100 rpm were compared with their tissue-flask and static–Petri dish controls. An expanding ESC population could be maintained, in suspension as aggregates, although the extent of cell growth was less than that achieved in tissue-culture flasks and Petri dish cultures (Figs. 2A, 2B). At 60 rpm, aggregates had larger and more variable sizes (CCE: 211.1 ± 89.2 μm; R1: 197.0 ± 98.0 μm) (Fig. 2H). In terms of cell growth, CCE suspension aggregates acquired a longer doubling time and were variable from trial to trial, resulting in the large standard deviation. Consequently, CCE cells expanded only 32.5 ± 22.8 times, while R1 cells expanded 25.2 ± 8.8 times, translating to a population doubling time of 32.1 ± 10.2 hours. To mediate the large variability in cell growth observed for our stirred-suspension culture system at 60 rpm, the agitation rate was increased to 100 rpm. The sizes of the resulting ESC aggregates were smaller and more uniform than those at 60 rpm (CCE: 136.2 ± 40.3 μm; R1: 116.5 ± 38.1 μm) (Fig. 2H). The population doubling times recorded for CCE and R1 cells were 23.5 ± 5.8 hours and 39.4 ± 19.4 hours, respectively. CCE cell aggregates cultured at 100 rpm expanded 53.4 ± 9.6 times in 15 days, as compared with 20.4 ± 11.0 times for R1 cells. Petri dish controls for both cell lines yielded 75.9 ± 17.4– and 49.0 ± 11.5–fold expansions for CCE and R1 cells, respectively.% D5 D# P' }1 `3 t" ?
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Figure 2. Undifferentiated ESCs could be cultured as suspension aggregates for an extended period of time via serial passaging. (A): Net population doubling times for CCE (filled bars) and R1 (hollow bars) cells cultured on tissue-culture flask controls, Petri dishes, and in spinner vessels agitated at 60 rpm and 100 rpm. Data are expressed as mean of three independent experiments ± SD. (B): Cumulative cell-fold expansions for CCE (filled bars) and R1 (hollow bars) cells cultured on tissue-flask controls, Petri dishes, and in spinner vessels agitated at 60 rpm and 100 rpm. Data are expressed as mean of three independent experiments ± SD. (C): Fifteen-day average of percentage of positive SSEA-1 expression detected in viable CCE (filled bars) and R1 (hollow bars) cells harvested from tissue-flask control, Petri dishes, as well as 60 rpm and 100 rpm spinner cultures. Data are expressed as mean of at least two independent experiments ± SD. (D): Fifteen-day average of percentage of positive E-cadherin expression detected in viable CCE (filled bars) and R1 (hollow bars) cells harvested from tissue-flask control, Petri dishes, as well as 60 rpm and 100 rpm spinner cultures. Data are expressed as mean of at least two independent experiments ± SD. (E): Oct-4 expression was quantified on each day of harvest plus on days of EB analyses for tissue cells obtained from () tissue-flask control, () Petri dish, () 60 rpm spinner flask, and () 100 rpm spinner flask. Data are expressed as mean of at least two independent experiments ± SD. (F): Representative dot plots of ESCs cultured using the three techniques and stained for Flk-1 and CD34 on day 15 of undifferentiated culture as well as day 4 and 9 of EB formation. (G): Reverse transcription–polymerase chain reaction analysis of ESCs and differentiation marker expression on day 15 of ESC culture and day 4 and day 9 of EB culture. (H): Morphology of R1 ESC suspension aggregates agitated at (i) 60 rpm and (ii) 100 rpm; images were obtained at x 40 magnification. Abbreviations: EB, embryoid body; ESC, embryonic stem cell.6 {/ g5 x0 t0 t& m7 v1 g' R

Importantly, although the population growth of the suspension ESC aggregates was lower than the controls, SSEA-1 and E-cadherin expression was >80% for both cell lines throughout the entire suspension culture period (Figs. 2C and 2D, respectively). Oct-4 also maintained high expression level during the culture period, along with subsequent downregulation upon EB formation (Fig. 2E). Flow cytometric analysis of Flk-1 and CD34 yielded expected expression patterns (Fig. 2F). RT-PCR analysis of Flk-1, HNF3-?, MHC, and Sox-1 gene expression was also consistent with normal ESC development (Fig. 2G).- [+ R9 R9 b! U  X

E-Cadherin Mediated the Extent of Cell–Cell Adhesion in Microcarrier and Suspension Aggregate Cultures
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We speculated that this high level of bead-bridging observed in our microcarrier cultures could be due to the expression of the cell adhesion molecule E-cadherin on undifferentiated ESCs . Microcarrier cultures performed using the M8 ESCs heterozygous for E-cadherin behaved similarly to CCE and R1 cells in microcarrier cultures (Fig. 3A). In contrast, similar cultures of the E-cadherin–null 9J ESCs showed that bead-bridging was dramatically reduced (Fig. 3B). A monolayer of 9J cells was able to attach onto the glass microcarrier surface; however, the cells did not spread out and flatten on the substratum, and most of the cells remained unattached, contributing to poor culture viability.& Z1 d2 E( C* p

Figure 3. Morphology of (A) E-cadherin /– M8 and (B) E-cadherin–/– 9J cells grown on glass microcarriers. M8 (C) and 9J (D) embryonic stem cell aggregate cultures established in spinner vessels agitated at 100 rpm. The cells were cultured in the same manner as the CCE and R1 microcarrier cultures (see text). Single cells were seeded into 100-ml spinner vessels and cultured for 3 days. 9J cells failed to formed aggregates spontaneously, whereas M8 cells generated smaller aggregates compared with CCE and R1 cells. The lack of E-cadherin expression caused 9J cells to remain mostly in single-cell suspension, with the occasional formation of large, irregularly shaped cell clumps, which led to high cell death (data not shown). Images (A) and (B) were obtained at x 100 magnification; (C) and (D) were obtained at x 40 magnification.& y# A; q. ~# F4 G# r( [
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M8E-cadherin /– ESCs were also cultured as suspension aggregates agitated at 100 rpm for 6 days with dissociation and reseeding on day 3. The average aggregate size was significantly smaller than those of CCE and R1 cells (M8: 69.2 ± 18.1 μm) (Fig. 3C). In contrast, 9J E-cadherin–/– ESCs were unable to form aggregates in suspension at 100 rpm. The culture consisted of mostly singly suspended cells with poor viability. There were also a small number of larger, irregularly shaped cell clumps that were formed (Fig. 3D).. @9 r  m" k; J) T5 c; T
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ESC Aggregates Could Be Induced to Form EBs in Differentiation Medium While Being Maintained in Stirred-Suspension Cultures; J  j' v* B% y/ c* u4 H( g

We asked next if a single-step expansion differentiation bioprocess could be developed based upon the shear-controlled expansion of ESCs. In this study, 50-ml suspension aggregate cultures agitated at 100 rpm were established, as described above, for the expansion of undifferentiated cells. After extended culture, LIF was removed from the media and the ESC aggregates were induced (at 100 rpm) to differentiate into EBs over 9 days. Analyses of cells generated from the EBs formed from the suspension-expanded ESC aggregates revealed that the cells exhibited normal differentiation kinetics. Oct-4, Flk-1, and CD34 had an expression pattern similar to that of the other culture techniques (Figs. 4A, 4B). In addition, phenotypic marker expression was also detected by RT-PCR as previously mentioned (data not shown). The nondissociated aggregates were able to form EBs that increased in size with time (Fig. 4C) in 100-ml suspension without detrimental agglomeration (CCE: day 4 EBs, 6,150 ± 1,485 aggregates; day 8 EBs, 2,600 ± 1,273 aggregates).# \4 z3 F' v+ H/ U9 S. v

Figure 4. ESC aggregates were successfully induced to form EBs without dissociation in stirred-suspension agitated at 100 rpm. One-ml aliquot of the culture was extracted from the spinner vessel and was placed in a 35-mm Petri dish. Dot plots generated from flow cyto-metric analysis of cells harvested from EBs demonstrate expected expression patterns for (A) Oct-4 and (B) Flk-1/CD34. (C): Images of suspension ESC aggregates, day 4 and day 9 EBs were taken at x 40 magnification. (D): CFC assay performed on differentiated cells generated from R1 ESC aggregates demonstrated the presence of colonies composed of cell types of the blood lineage such as (i) erythrocytes and (ii) granulocytes. Images were taken at x 100 magnification. Abbreviations: CFC, colony-forming cell; EB, embryoid body; ESC, embryonic stem cell.

To demonstrate that the ESC suspension aggregates were able differentiate into functional cardiac and hematopoietic cells, expanded aggregates were induced to differentiate upon LIF removal and the resultant EBs were tested for their ability to form contractile areas and hematopoietic CFCs, respectively, using standard assays (see Materials and Methods). Beating areas of cells were formed from these EBs, as expected, after attachment (data not shown). Using the CFC assay, we observed that cells dissociated from EBs generated with ESC aggregates generated both erythroid and myleomonocytic colonies (Fig. 4D). In both cases, no detectable differences relative to standard control conditions were observed.% r8 ]* U: U+ @4 F* l( O

DISCUSSION5 [4 J+ g2 m& y# r; s* m' t3 s

The authors would like to thank Amersham Biosciences and Dr. William Hillegas from SoloHill Engineering, Inc., for providing advice and reagents associated with the microcarrier culture systems. We also appreciated the provision of the M8 and 9J murine ESC lines from Dr. Lionel Larue (Institut Curie, France). This work was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the National Science Foundation (NSF) through an Engineering Research Center (ERC) grant to the Bioprocess Engineering Research Center (BPEC) at the Massachusetts Institute of Technology. P.W.Z. is the Canada Research Chair in Stem Cell Bioengineering.$ n/ i" q- D( p. V: T

DISCLOSURES

The authors indicate no potential conflicts on interest.

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