Laboratory of BioProcess Systems Engineering
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan

Laboratory of BioProcess Systems Engineering (BPSE) has been organized by Prof. Kino-oka
since April 2009. The laboratory mission is to bring a good fortune in human life through theelucidation and utilization of “bio-potential” by understanding sequential biological events(BioProcess) in the reaction field (Systems). The main stream in BPSE, as shown in Fig.1, isthe bioprocess designs in stem cell and tissue engineering. The research interests, which aretargeted for the biological elements governing the reactions (ie. cellular differentiation,
undifferentiation, de-undifferentiation, maturation, tissue self-assembling, functionalizationetc.) as well as the regions providing the reaction field (ie. 2-D colonies and 3-D spherical andsheet aggregates), are to establish methodologies for process and quality controls in
bio-production including the analysis, simulation, forecasting, making great contribution to thestem cell industry (ie. the outputs for manufacturing transplants through design of process andquality controls, for drug screening systems through development of bio-mimic structure, andfor mass productions of human biologics through design of scale-up). The outcome includes theestablishment of manufacturing process & quality controls (ie. culture design, bioreactor
development, monitoring, process automation, etc.), the development of human resources asbiochemical engineers, and establishment of standardizations and guidelines for stem cellindustrialization.
BPSE were organized with four groups, (A) stem cell group for the maintenance of
undifferentiation to prevent de-undifferentiation and for the directional regulation ofdifferentiation, (B) cell expansion group for designs of culture process and intelligent

Fig.1 Mission of BPSE

automation system, (C) functional 3-D tissue group for vascular network formation a Local
habitat distribution in multilayered cell sheet and mechanical strength in cultured tissue, and(D) culture simulation group for process and quality controls as an interdisciplinary technology
supported by the above academic fields.

A. Stem Cell group
The main topics in this group are themethodological development for
regulation of cytoskeletal signaling(Rho GTPase family of Rac1, cdc42,
RhoA) by changing the cellularmorphology with dynamic behaviors
on the glucose-displaying dendrimer
surface as shown in Fig.2.

Fig.2 Availability of D-glucose displaying dendrimer
surfaces

BPSE proposed a possible
mechanism underlying the cellanchoring and morphologicalchanges in cultures of humanepithelial
cellsonD-glucose-displaying
surfaces(1-8). The variation in the ratio of
D-glucose
displayedcausedcellular morphological changes
that depended on the presence orabsence of insulin. In addition, Fig.3 Directional regulation of stem cells based on the
fluorescencemicroscopyofregulation of Rho GTPase family
F-actin, vinculin, and GLUTsclarified the localization of
integrin-mediated as well as GLUT-mediated anchoring, leading to the consideration that themorphological changes of cells are responsible for the variation in the balance between thequantities of D-glucose on the culture surface and GLUTs on the cytoplasmic membrane, whichis associated with the promotion of focal contact formation mediated by GLUTs. The review
article (6) mentioned that the designs using the surfaces are based on the novel cell binding
mechanism in which GLUTs are key elements responsible for regulating cell attachment andmorphologies as well as development of the cellular phenotypes.
Although specific ligand-receptor interactions on solid surfaces are important for regulatingcell shapes in various cell types, the properties of the surfaces also play key roles indetermining the cellular functions and fates. Identifying specific cues in microenvironment andunderstanding how neighboring cells and ECM control developmental fates will be required topromote the differentiation of cells, such as stem cells, into targeted cell lineages. BPSE
proposed the idea for cytodifferentiation of stem cells accompanied by the regulation ofcytoskeletal formation through cell morphological variation as well as the intracellular signaltransduction through cell aggregate formation (Fig.3). BPSE recently reported that thealterations in Rho family GTPase activities derived from cytoskeletal formation can lead toguidanceofcardiomyogenic
differentiationof
human
mesenchymal stem cells (hMSCs)
during in vitro culture (8). To
regulate the cytoskeletal formation of
hMSCs, a dendrimer-immobilized
substrate that displayed D-glucose
was employed (Fig.4). With anincrease in the dendrimer generation
number, the cells exhibited active
migration, accompanied by cellmorphological changes of stretchingandcontracting
withoutany
modification of culture media. On
the 5th-generation dendrimer surface,in particular, the cells exhibited Fig.4 Cardiomyogenic differentiation capacities of
RhoA down-regulation and Rac1 hMSCs (7 days)
up-regulation during the culture, associated with alterations in the cellular morphology andmigratory behaviors. It was also found that cell aggregate formation was promoted on thissurface, supporting the notion that an increase in N-cadherin-mediated cell-cell contacts andWnt pathway signaling regulate hMSC differentiation into cardiomyocyte-like cells. Thissuggests that the changes in the cytoskeletal organization have a direct impact on the
cardiomyogenic differentiation of hMSCs in relation to Rho family GTPase-dependent
signaling pathways. Thus, the transporter-mediating anchoring (D-glucose displaying on thesurface) is one of the powerful techniques to realize the regulation of the cellular morphology
accompanied by the cytoskeletal variation, leading to commitment of stem cells to different
lineages. This technique currently applied to the maintenance culture of human iPS cells andcan make the directional control to stay undifferentiated state of iPS cells without any
derivation to de-undifferentiated state.

2. Cell Expansion group
Bioreactors are a core element to produce high-value materials in biological processes using
animal cells. In the cultures of animal cells, the major features are the mechanical fragility
and low growth rate of these cells, which leads to the strategy of high cell density culture by
maintaining the cells within bioreactors with an external medium flow. In recent decades,
technologies for cell and tissue therapies have emerged in the field of regenerative medicine.
For such purposes, the targets are ex vivo expansion of human cells in undifferentiated ordifferentiated states, where the products of interest are cells and/or tissue themselves. This is an
emerging interdisciplinary area of research and technology development that has the potential
to revolutionize methods in medicine and leads to the requirement for small-scale design ofpatient-oriented bioreactors for clinical use. In addition, the bioreactor used in these cultures
should be operated under strictly controlled conditions to produce cells or tissues with quality
and functions that meet specific clinical requirements. BPSE has developed the automation for
expansion process including the operations of seeding, medium change, passage as well as
observation(9,10), and proposed the intelligent culture system accompanied by automatedoperations (liquid transfer and cell passage)
to perform serial cultures of human skeletal
muscle myoblasts, as shown in Fig.5 (11).
An automated culture system that could
manage two serial cultures by monitoring
the confluence degree was constructed. The Fig.5 Intelligent bioreactor system for passage
automated operation with the intelligent automation
determination of the time for passage was
successfully performed without serious lossof growth activity, compared with manual
operation using conventional flasks. This
intelligent culture system can be applied to
cultures of other adherent cells and will leadto the qualitative stability of products in thepractical manufacturing of cells available fortransplantation. In other words, the culture
system will contribute to the process control
as well as the quality control. Recently, thistechnique applied to the development of chip
culture system for maturation of retina
pigment epithelial cells derived from human Fig.6 Chip bioreactor system for maturation of
iPS cells(Fig.6).
retina pigment epithelial cells
Innovative techniques of cell and tissue
processing, based on tissue engineering, have been developed for therapeutic applications. Cell
expansion and tissue reconstruction through ex vivo cultures are core processes used to produce
engineered tissues with sufficient structural integrity and functionality. In manufacturing, strict
management against contamination and human error is compelled due to direct use of
un-sterilable products and the laboriousness of culture operations, respectively. Therefore, thedevelopment of processing systems for cell and tissue cultures is one of the critical issues for
ensuring a stable process and quality of therapeutic products. However, the siting criterion ofculture systems to date has not been made clear. BPSE review article classifies some of the
known processing systems into ‘sealed-chamber’ and ‘sealed-vessel’ culture systems based on
the difference in their aseptic spaces, and describes the potential advantages of these systems
and current states of culture systems (12). Moreover, on the basis of the guidelines for isolator
systems used in aseptic processing for healthcare products, which are issued by theInternational Organization for Standardization, the siting criterion of the processing systems forcells and tissue cultures is discussed in perspective of manufacturing therapeutic products inconsideration of the regulations according to the Good Manufacturing Practice.
On the basis of the siting criterion, the comparison of management between cell processing
facility (CPF) and cell aseptic processing system (CAPS) based on the isolator system revealed
that CAPS leads to reductions of the running cost as well as operational laboriousness in thesmall production. Recently, BPSE, as shown in Fig.7, proposed a novel design ofmanufacturing facility based on a flexible Modular Platform (fMP) which will reach thecompactness of aseptic processing area and quick change-over for multi-purposes and patients,
leading to cost-saving with safety and security .

As shown in Fig.8,
BPSE collaborates with
Profs. Okano and Shimizu
inTokyo
Women’s
Medical University andseveral companies todeveloptheisolatorsystem for the sheet
assembly based on thefMP, which will makeFig.8 Automation system of sheet assembly based on the fMP
automated formation ofmultilayered sheets andtheir
incubation.
Themachinery operations weresuccessfully performed. And
this system can realize someprocedures by having flexible
connections
withvariousmodules required for theculture operations under sterile
conditions, suggesting thebroad versatility for the
production in other types ofmultilayered sheets.
Fig.7 Proposal of manufacturing system based on flexible
Modular Platform (fMP)
3. Functional 3-D tissue group
The innovative techniques based on tissue engineering have been developed, being now at apractical stage of clinical application by means of grafting in vitro cultured products. The
representative process of autologous production of cultured tissues, which consists mainly ofplanar (monolayer) growth for cell expansion and spatial growth for tissue functioning. So far,a variety of natural and artificial materials available for scaffolds, which permit target cells to
make aggregation, have been designed for the spatial growth and differentiation of the cells.
The spatial growth in a scaffold leads to distribution in cell density owing to limitation ofnutrient supply, causing the variation in quality of cultured tissues (spatial heterogeneity). In
addition, another core process for cures using cells is the subculture for cell expansion to
produce tissue-engineered tissues. However, the monolayer growth of target cells often causes
the dedifferentiation accompanied with morphological variation (population heterogeneity),
causing insufficient quality of cultured tissues. Therefore, it is necessary to evaluate thepopulation heterogeneity as an indicator for state of differentiation during the subcultures, andto estimate spatial distribution of cell aggregate in cultured tissues. These techniques will becontributable to QC especially for efficacy of cultured tissues (13).
Many techniques for the QC were developed mainly for safety of cultured tissues. From aviewpoint of engineering, therefore, the strategic construction to estimate the QC for efficacy isinevitable. BPSE, as shown in Fig.9, has focused on the tool development for spatial estimationof cells in cultured tissues(14-16).

The stereoscopic image
analysis of fluorescence-labeledchondrocyte cells for cytoplasm
and nucleus was performed forthe quantitative determinationof spatial cell distribution as
well as cell aggregate size in thecollagen-embedded culture. The
three-dimensional
histomorphometricdata
indicated that the cells in thegels formed aggregates by cell
division, and the size ofaggregatesincreasedwithelapsed culture time, revealing
the mechanism of the spatial
heterogeneityincell Fig.9 Experimental procedures with spatial cell
distribution.
Further distribution analyzer consisting of image capturing and its
development describes the processing
morphological assessment ofchondrogenic potency during acell expanding process throughserial subculturing, although themonolayer
growthofchondrocytes
causesdedifferentiation and results in a
dysfunctional cartilage structure
after transplantation, and thefrequency of spherical-shaped
cells in the CL gels was found to
decreasealong
withdedifferentiationwhen
thepassaged cells were embedded in Fig.10 Experimental procedure for analyzing spatial cell
the CL gels suggesting that the nucleus distribution by means of image capturing andmorphological evaluation enables processing, and definition of basal layer in multilayer
the grasping the chondrogenic sheet
potency in terms of thephenotypes and differentiate
states, ultimately leading to quality control of tissue-engineered cartilage. This imaging
technique, as shown in Fig.10, was applied to estimate the densities of total and proliferative
cells by staining of whole nuclei and proliferative nuclei, respectively in 3-D epithelial sheets(17).
The cell sheet engineering is emerging as an advanced technique with preparation ofscaffold-free three-dimensional (3-D) tissue, not only for transplantations, but also for in vitro
researches.
Recently autologous transplantation of multilayered myoblast sheet has been
attracting attention as a new technique for curing myocardial infarction, which is associatedwith the dysfunction of cardiomyocytes and irreversible cell loss. This method can overcome
the disadvantages such as less take ratio of transplanted cells through the direct injection ofmyoblast suspension. Skeletal myoblasts, which are easy to be harvested from patients, haveability to become active, self-renew and differentiate, permitting muscle regeneration uponmuscle injury. As shown in Fig.11, the sheet of myoblasts also has ability to source thecytokines which improve heart function due to paracrine system including the facilitation ofangiogenesis and the attraction of progenitors on affected part. From manufacturing point ofview, the system development for process and quality controls is important to be concerned,
leading to the active commercialization using the cell sheets. Many researches have beentackled concerning cell source exploring, cell culture, sheet assembling, and in vivo animal tests.
However, the method for quality control of myoblast sheet, especially for transplant efficacy,
has not been systematized. The image analysis technique described above was applied to theculture system that uses multilayered sheet containing stained target cells in the basal layer andconfocal laser scanning microscopy, and realizes clear observation of target cell behaviors in
the vertical direction, enabling mono-dimensional analysis of vertical cell distribution inside
the sheet (18). The reduced spatial
dimension makes easy to analyze cell
migration, compared to the full 3-D
analysis required of sphericallyshaped aggregates. Thus, the systemdeveloped in the present study can bea powerful tool for elucidating
dynamic
phenomenain
3-D
constructs.
Five-layered myoblast sheet was
fabricated as a 3-D model to evaluate
vertical cell migration by confocal
laser scanning microscopy withimage processing. And BPSE
establishes the mimic system oftransplantation which consists ofendothelial cells (HUVECs) on
culture dish as target cells on lesion
site and five-layered myoblast sheet
as transplants, and focus onunderstanding
angiogenesis
procedure as post transplantation.
The proposed mimic system, as
shown in Fig.12, expresses theformation of endothelial network
formationandpossesses
theoperational variables such as
incubation time, initial density of
HUVEC, myoblast density in sheet,contamination population of skeletal
fibroblasts and so on. The populationof skeletal myoblasts and fibroblasts,

Fig.11 Conceptual drawing of new approach for
quality evaluations of multilayer sheet throughangiogenesis

Fig.12 Profile of network formation of HUVEC in
multilayered sheetwhich depends on cell sources from the patients, drastically affected the HUVEC connectivity.
These results suggest our mimic system can make in vitro quantitative estimation ofangiogenesis potential for the transplants from the patients.

4. Culture Simulation group
In manufacturing, the “raw materials” of cells
isolated from patients (or donors) are ofheterogeneity in cell states, and the “products” ofcultured tissues have variation in the required
volume for individual cultured tissues (products).
During the three-dimensional culture, the spatial
Fig.13 Spatial cell distribution in
distribution and aggregation of target cells in thecultured cartilage at 21 days
cultured constructs is considered to occur owing to
the limitation of oxygen and protein supplies. In
addition, from a practical standpoint of cultured tissue production, the extent of cell divisionthat depends on the states of origins such as donor age and location of harvesting biopsy
becomes an obstacle to acquisition of sufficient number of cells in the 3-D cultures. The
heterogeneity found in the culture process leads to the batch process with less reproducibility.
These imply the production in a so-called “tailor-made process” where culture operations mustbe handled case by case. Therefore, the reliable and robust process is required for
manufacturing the products with high quality while considering the instability and fluctuationin conditions of the raw materials.
The spatial distribution of target cells in the construct is very important information forevaluation of cultured tissues and it is desired to estimate the potentials of population doublingsin the 3-D cultures. In particular, a feasible method to evaluate culture states is needed tosupport the operators who have to judge the cessation of 3-D cultures with useful information.
To analyze the growth manner, a kinetic model is a promising tool for the prediction of spatial
heterogeneity which affects the quality of cultured tissues (19).
BPSE proposed the stochastic modeling based on cellular automaton which can be introduced
to express the complex dynamics of cell proliferation in the construct (19-20). As shown in
Fig.13, this model revealed that the special distribution of cell aggregates depends on theculture time and cell seeding density. The profiles of cell number and aggregate distribution are
attributed to the limitation of oxygen supply as well as spatial contact inhibition due to the lossof space capable for cell division, similarly to those derived from the observation data, being a
useful tool to estimate the overall cell propagation and spatial cell distribution in thecollagen-gel embedded culture of chondrocytes.

Selected References
1. M. Kino-oka, Y. Morinaga, M.-H. Kim, Y. Takezawa, M. Kawase, K.Yagi, M. Taya:“Morphological regulation of rabbit chondrocytes on glucose-displayed surface”,
Biomaterials, 28, 1680 -1688 (2007).

2. M.-H. Kim, M. Kino-oka, M. Kawase, K. Yagi, M. Taya: “Synergistic effect of
D-glucose and EGF display on dynamic behaviors of human epithelial cells”, J. Biosci.
Bioeng., 104, 428-431(2007).
3. M.-H. Kim, M. Kino-oka, M. Kawase, K. Yagi, M. Taya: “Glucose transporter
mediation responsible for morphological changes of human epithelial cells on
glucose-displayed surfaces”, J. Biosci. Bioeng., 105,319-326 (2008).
4. S. Mashayekhana, M.-H Kim, S. Miyazaki, F. Tashiro, M. Kino-oka, M. Taya, J.
Miyazaki: “Enrichment of undifferentiated mouse embryonic stem cells on a culture
surface with a glucose-displaying dendrimer”，Biomaterials, 29, 4236–4243 (2008).
5. M.-H. Kim, M. Kino-oka, Y. Morinaga, Y. Sawada, M. Kawase, K. Yagi, M. Taya:“Morphological regulation and aggregate formation of rabbit chondrocytes on
dendrimer-immobilized surface with D-glucose display”, J. Biosci. Bioeng., 107,
196-205 (2009).
6. M.-H. Kim, M. Kino-oka, M. Taya: “Designing culture surfaces based on cell anchoringmechanisms to regulate cell morphologies and functions”, Biotechnol. Adv., 28, 7–16
(2010).
7. M.-H. Kim, M. Kino-oka, A. Saito, Y. Sawa, M. Taya: “Myogenic induction of humanmesenchymal stem cells by culture on dendrimer-immobilized surface with D-glucose
display”, J. Biosci. Bioeng., 109, 55–61 (2010).
8. M.-H. Kim, M. Kino-oka, N. Maruyama, A. Saito, Y. Sawa, M. Taya: “Cardiomyogenic
induction of human mesenchymal stem cells by altered Rho family GTPase expression
on dendrimer-immobilized surface with D-glucose display”, Biomaterials, 31,
7666-7677 (2010).
9. M. Kino-oka, J. E. Prenosil: “Development of on-line monitoring system of humankeratinocyte growth by image analysis and its application to bioreactor culture”,
Biotechnol. Bioeng., 67, 234-239 (2000).
10. M. Kino-oka, N. Ogawa, R. Umegaki, M. Taya: “Bioreactor design for successive
culture of anchorage-dependent cells operated in an automated manner”, Tissue Eng.,11, 535-545 (2005).
11. M. Kino-oka, S. R. Chowdhury, Y. Muneyuki, M. Manabe, A. Saito, Y. Sawa, M. Taya:“Automating the expansion process of human skeletal muscle myoblasts with
suppression of myotube formation”, Tissue Eng., 15, 717-728 (2009).
12. M. Kino-oka, M. Taya: “Recent developments in processing systems for cell and tissue
cultures toward therapeutic application”, J. Biosci. Bioeng., 108, 267–276 (2009).
13. M. Kino-oka, Y. Takezawa, M. Taya: “Quality control of cultured tissues requires tools
for quantitative analyses of heterogeneous features developed in manufacturing
process”, Cell and Tissue Bank, 10, 63-74 (2009).
14. M. Kino-oka, Y. Maeda, Y. Sato, A. B. Khoshfetrat, T. Yamamoto, K. Sugawara, M.
Taya: “Characterization of spatial growth and distribution of chondrocyte cells
embedded in collagen gels through a stereoscopic cell imaging system”, Biotechnol.
Bioeng., 99, 1230-1240 (2008).

15. A. B. Khoshfetrat, M. Kino-oka, Y. Takezawa, T. Yamamoto, K. Sugawara, M. Taya:“Seeding density modulates migration and morphology of rabbit chondrocytes cultured
in collagen gels”, Biotechnol. Bioeng., 102, 294-302 (2009).
16. M. Kino-oka, Y. Maeda, Y. Sato, N. Maruyama, Y. Takezawa, A. B. Khoshfetrat, K.
Sugawara, M. Taya: “Morphological evaluation of chondrogenic potency in passaged
cell populations”, J. Biosci. Bioeng., 107, 544-551 (2009).
17. M.-H. Kim, N. Tsubakino, S. Kagita, M. Taya, M. Kino-oka：“Characterization of
spatial cell distribution in multilayer sheet of human keratinocytes through astereoscopic cell imaging system”, J. Biosci. Bioeng., 112, 289-291 (2011).
18. M. Kino-oka, T. X. Ngo, E. Nagamori, Y. Takezawa, Y. Miyake, Y. Sawa, A. Saito, T.
Shimizu, T. Okano, M. Taya: “Evaluation of vertical cell fluidity in a multilayered sheetof skeletal myoblasts”, J. Biosci. Bioeng., 113, 128-131 (2012).
19. M. Kino-oka, Y. Maeda, T. Yamamoto, K. Sugawara, M. Taya: “A kinetic modeling ofchondrocyte culture for manufacture of tissue-engineered cartilage”, J. Biosci. Bioeng.,
99, 197-207 (2005).
20. M. Kino-oka, R. Umegaki, M. Taya, S. Tone, J. E. Prenosil: “Valuation of growthparameters in monolayer keratinocyte culture based on a two-dimensional cell
placement model”, J. Biosci. Bioeng., 89, 285-287 (2000).