Plant cell culture technology in the cosmetics and food industries: current state and future trends

Abstract

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.

Full text

MINI-REVIEW
Plant cell culture technology in the cosmetics and food industries:
current state and future trends
Regine Eibl
1
&Philipp Meier
1
&Irène Stutz
1
&David Schildberger
2
&Tilo Hühn
2
&Dieter Eibl
1
Received: 12 June 2018 / R evised: 27 July 2018 / Accepted: 28 July 2018 /Pu blished online: 11 August 2018
#
Abstract
The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The
emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in
plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made
their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In
addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced
at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell
culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition,
two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts
health and food that can be produced in the lab or at home.
Keywords Bioreactor .Cellular agriculture .Cosmetic supplement ingredients .Foodstuff and foodingredients .Plan t cell culture
extracts
Introduction
In 1902, the Australian botanist Gottlieb Haberlandt provided
the basis for the usage of plant cell and tissue cultures
(Haberlandt 1902). He described the formation of callus (un-
organized cell mass in response to wounding) from adult plant
cells and its regeneration into a complete plant for the first
time. This phenomenon, also known as cellular totipotency
of plant cells, was experimentally demonstrated by growing
carrot cells in vitro by Haberlandt in 1958 (Fehér 2015).
Between the 1960s and the 1980s, many studies were execut-
ed in order to mass propagate plant cell cultures and to devel-
op bioprocesses delivering secondary metabolites for the phar-
maceutical, food, and cosmetics industries. Different
commercial secondary metabolites (e.g., shikonin, scopol-
amine, protoberines, rosmarinic acid, ginseng saponins, and
immunostimulating polysaccharides), which are based on
plant cell cultures, entered the market between the early
1980s and late 1990s (Sato and Yamada 1984;Denoetal.
1987; Ritterhaus et al. 1990;Hess1992; Hibino and
Ushiyama 1999). A further milestone in plant cell culture
technology is represented by the FDA (Food and Drug
Administration) approval of the anticancer compound pacli-
taxel in 2000. Cells from the Pacific yew grown in 75 m
3
stirred bioreactors deliver up to 500 kg of this medicinally
important secondary metabolite per year (Imseng et al. 2014;
Steingroewer 2016). The advantages of the production of sec-
ondary metabolites with plant cell cultures over conventional
agricultural production with whole plants are indisputable
(Hussain et al. 2012). There is no seasonal dependence on in
vitro production of secondary metabolites, and a controlled
manufacture via standardized batches is possible.
Furthermore, the impact on the ecosystem is low, the water
needed and carbon footprint are reduced, and pesticides as
well as herbicides are not required. Nevertheless, the number
of commercial production processes of secondary metabolites
involving plant cell cultures is low. This particularly concerns
pharmaceutical applications and is ascribed to somaclonal
*Regine Eibl
eibs@zhaw.ch
1
Institute of Chemistry and Biotechnology, Zurich University of
Applied Sciences (ZHAW), 8820 Wädenswil, Switzerland
2
Institute for Food and Beverage Innovation, ZHAW,
8820 Wädenswil, Switzerland
Applied Microbiology and Biotechnology (2018) 102:8661–8675
https://doi.org/10.1007/s00253-018-9279-8
The Author(s) 2018
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variations of the production clones as well as too low second-
ary metabolite titers (Sharma et al. 2014).
Product approval in the pharmaceutical industry differs
from that in the cosmetics industry, where no official approval
is required and where the manufacturing company is respon-
sible for product safety (Zappelli et al. 2016). Moreover, in-
novations and developments in the cosmetics industry, which
introduces hundreds of new cosmetics products every year,
are strongly driven by the consumer. The consumer wants to
have not only effective, safe, and natural but also sustainable,
cosmetics products, whose manufacture does not negatively
affect the environment (Schmidt 2012;Fonseca-Santosetal.
2015). In respect of the cosmetics industry, there is high inter-
est in plant cell culture extracts with multiple specific activities
for skin care, make-up, and hair care as supplement ingredi-
ents. Plant cell culture extracts containing a mixture of bioac-
tive ingredients (and not only secondary metabolites) can al-
ready be produced under controlled conditions. Moreover,
even extracts from rare or endangered plant species can be
made available by applying plant cell culture technology. It
is also worth mentioning that plant cell culture extracts can be
used in minimal concentrations in the final cosmetics formu-
lations (Barbulova et al. 2014). In other words, a low product
titer is less critical than in pharmaceutical applications, espe-
cially since the plant cell culture extract may act in a syner-
gistic manner as described by Carola et al. (Carola et al. 2012).
Consequently, the large number of cosmetics products which
have been manufactured with plant cell culture technology
over the past 10 years is hardly a surprise. Indeed, it explains
the renaissance in plant cell culture technology that has taken
place.
The developments in the cosmetics industry have influ-
enced the food industry, where new manufacturing methods
for food and food ingredients are also in demand. Various
studies havereported that supplying the world population with
both animal and plant-based food in sufficient quantity and
quality will become increasingly difficult. For example, ac-
cording to the estimates of Alexandratos and Bruinsma, 60%
more food will be required by 2050 than is manufactured
today (Alexandratos and Bruinsma 2012), and traditional
farming will not be able to meet these requirements. Cellular
agriculture is assumed to be one solution here (Foussat and
Canteneur 2016;Mattick2018; Nordlund et al. 2018). Plant
cell-based cellular agriculture uses plant cell cultures to man-
ufacture high-value food ingredients (Stafford 1991;Fuetal.
1999; Ravishankar et al. 2007; Nosov 2012;Daviesand
Deroles 2014). Ginseng triterpene saponins manufactured
with plant cell cultures in bioreactors have been used as food
supplement ingredients for a considerable time (Wu and
Zhong 1999; Sivakumar et al. 2005;Paeketal.2009), but
many plant cell culture lines producing food supplement in-
gredients have not reached commercial production. Due to the
latest approaches to engineer homogeneous and high-
productivity cell lines without genetic engineering (Yun et
al. 2012; Sood 2017), plant cell culture technology for food
products is regaining interest. Climate change and plant dis-
eases reducing the production of plant-based food are driving
this trend, and first scientific studies have suggested that plant
cell cultures or their extracts may themselves be used as food-
stuffs (Räty 2017;Nordlundetal.2018).
This mini review describes the current state of plant cell
culture technology aimed at products for the cosmetics and
food industries. Based on an overview of plant cell culture
extracts which have been launchedby European and US com-
panies over the past 10 years, we present the main plant cell
culture types of interest, their establishment, the mass propa-
gation in bioreactors, and the related challenges. In addition,
the production of plant cell culture extracts following the bio-
reactor cultivation step is briefly discussed. Finally, two ex-
amples of the latest developments in plant cell culture-based
food production are given. However, plant cell culture-based
manufacture of recombinant proteins (Tschofen et al. 2016)is
not covered, as the vast majority of cosmetics and food com-
panies, particularly those located in Europe, do not use genet-
ically modified plant cell and tissue cultures, which are the
precondition for the production of recombinant proteins with
plant cell culture technology.
Product overview of plant cell culture extracts
for applications in the cosmetics and food
industries
In 2008, Mibelle Biochemistry laid the cornerstone for the
successful course of plant stem cell culture extracts into the
cosmetics industry. The company launched
PhytoCELLTECH Malus domestica (Schmid et al. 2008;
Schürch et al. 2008;Imsengetal.2014), the first commercial-
ly available plant cell culture extract whose effect was studied
on human skin cells and which claims to be derived from plant
stem cells. PhytoCELLTECH Malus domestica was
established from the core of an endangered Swiss apple vari-
ety, the Uttwiler Spätlauber, which can be stored for a long
time without becoming shriveled or losing flavor. The com-
pany has patented the manufacture and usage of apple cell
culture extracts which originate from Malus domestica culti-
var Uttwiler Spätlauber and which protect skin cells (Blum et
al. 2013). The manufacture includes cell culture establish-
ment, their cultivation from shake flasks up to the production
bioreactor (50–100 L), and liposomal extract manufacture by
applying high pressure homogenization. PhytoCELLTECH
Malus domestica entails numerous plant cell culture extracts
which are used by leading cosmetics brands such as Dior,
Lancôme, Guerlain, and La Prairie in their cosmetic formula-
tions. The final products include facial serums, facial creams
and facial masks, eye creams, make-up products, hair oils, hair
8662 Appl Microbiol Biotechnol (2018) 102:8661–8675
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Table 1 Plant cell culture extracts manufactured by European and US companies which have entered the market in the cosmetics and food industries during the past 10 years. Asian manufacturers are not
considered. The manufacturer’s names are listed in alphabetic order (no rating). The list of products makes no claim to be complete
Product Plant species Application Manufacturer Reference
Acetos 10P Lippia citriodora Food: supplement ingredient Active BotanicalsResearch (ABR)
www.abres.it
Fremont (2017)
Teupol 10P and Teupol 50P Ajuga reptans
Echinan 4P Echinacea angustifolia
Celtosome Crithmum maritimum Cosmetics: skin rejuvenation
and care
BiotechMarine by Seppic
www.seppic.
com/seppic/biotechmarine
John (2017)
Eryngium maritimum
Cocovanol
1
Theobroma cacao Food: supplement ingredient Diana Plant Sciences
2
www.
diana-food.com;www.symrise.
com
Barney (2013), Georgiev (2015)
Plant C-Stem Vigna radiata Vigna radiata Cosmetics: skin rejuvenation
and care
innovacos
www.innovacos.com
Khan (2017)
Stem cell extracts from: roseroot,
greater plantain, milk thistle, Aloe
vera
Rhodiola rosea,Plantago major,
Silybum marianum,Aloe barbadensis
Mill.
In vitro Plant-tech
www.invitroplanttech.se
Bengtson (2018)
Flower Power Innovation extract Calendula officinalis and Silybum
marianum
Stems GX products: Buddleja Stems
GX, Echinaceae Stems GX,
Gardenia Stems GX, Lenontopod
Stems GX, Resistem, Marubium
Stems GX
Buddleja davidii,Echinacea
angustifolia,Gardenia jasminoides,
Leontopodium alpinum,Globularia
cordifolia,Marrubium vulgare
Institute of Biotechnological
research (IRB) by Sederma
3
www.sederma.fr); (www.irbtech.
com;www.croda.com
Dal Toso and Melandri (2010),
Schäfer (2012)
Stems GX products: Centella Stems
GX
Centella asiatica Cosmetics: treatment of rosacea Institute of Biotechnological
research (IRB) by Sederma
3
www.sederma.fr;www.irbtech.
com;www.croda.com
Dal Toso and Melandri (2011a),
Dal Toso and Melandri
(2011b), Unknown (2013)Dermasyr 10 Syringa vulgaris (lilac) Cosmetics: treatment of acne and
sebum-related disorders
Echigena plus Echinacea angustifolia Food: supplement ingredient
Teoside 10 Ajuga reptans
ReGeniStem Brightening Glycyrrhiza glabra Cosmetics: skin brightening Lonza
www.lonza.com
Lonza (2014)
Stem cell culture extract from arctic
cloudberries
Rubus chamaemorus Cosmetics: skin rejuvenation and
care
Lumene
www.lumene.com
Nohynek et al. (2014); Suvanto
et al. (2017)
PhytoCELLTECH actives from:
argan tree, apple, alpine rose,
soapwort, comfrey and grapes
Argania spinosa,Malus domestica
(Uttwiler Spätlauber), Rhododendron
hirsutum,Saponaria pumila,
Symphytum officinale,Vitis vinifera
(Gamay Teinturier Fréaux)
Mibelle Biochemistry
www.mibellebiochemistry.com
Schmid et al. (2008), Schmid and
Zülli (2012), Schmid et al.
(2013), Imseng et al. (2014),
Morus et al. (2014), Trehan et
al. (2017)
RootBioTec HO Ocimum basilicum Cosmetics: treatment of hair loss Belser (2015)
Callus stem cell extracts from:
orchid, lotus, tomato, rice, grape,
carrot, green tea, ginseng
Neofinetia falcata,Nelumbo nucifera,
Solanum lycopersicum,Oryza sativa,
Vitis vinifera,Daucus carota,
Camellia sinensis,Panax ginseng
Cosmetics: skin rejuvenation and
care
Sandream Enterprises
www.sandreamimpact.com
www.ultraspector.com. retrieved
on May 11, 2018
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serums, and hair conditioners. Table 1contains a selection of
plant cell culture extracts which are important for the cos-
metics industry and which contain, for example, polyphenols,
vitamins, fatty acids, peptide mixtures, and saccharides. They
have been successfully brought to market by European and
US companies over the past 10 years. The prevailing majority
of these product candidates have Bstem cell^in their product
name. It indicates that the plant cell culture extract originates
from plant meristems such as a shoot apical system, root api-
cal system, or cambium (Greb and Lohmann 2016). In Table
1, plant cell culture-based food supplement ingredients and
their manufacturers are also shown. Teoside 10 was the first
plant cell culture extract approved as a food supplement in-
gredient in Europe (Dal Toso and Melandri 2010;DalToso
and Melandri 2011a; Dal Toso and Melandri 2011b).
Recently, the food supplement ingredients Acetos 10P,
Teupol 10P, Teupol 50P, and Echinan 4P have been authorized
as Novel Food according to Article 5 of the EU Regulation
258/97 (Fremont 2017).
Applied plant cell culture types
Cell suspension cultures from dedifferentiated callus
cells and undifferentiated cambial meristematic cells
The plant cell culture extracts are most frequently derived
from cell suspension cultures that have been created from
dedifferentiated plant cells (DDCs). The common modus
operandi to establish a DDC-based plant cell suspension cul-
ture is illustrated in Fig. 1. The main steps of the procedure
include the selection of potent parent material, an optimized
surface sterilization procedure, the induction, maintenance
and mass propagation of the callus culture in petri dishes,
the initiation, homogenization, maintenance and mass propa-
gation of the suspension culture in shake flasks and bioreac-
tors, and the final cell banking of the suspension production
cell line. Although all parts of a plant can be used to initiate a
callus culture, it is important to select the most suitable parent
plant and organ type which contains the bioactive com-
pound(s) of interest in the desired quantity and quality. The
quantity and quality of the bioactive compound(s) of interest
are greatly affected by the plant species, its development stage
and location, and the plant organ (also referred to as explant)
type. Growth regulators (phytohormones which are a combi-
nation of auxins and cytokinins) that are added to the culture
medium also have to be taken into account when establishing
a high performing callus culture that is friable, grows and
produces well, and is stable. Both type and concentration of
the growth regulators have an influence on callus growth and
morphology as well as on secondary metabolite synthesis
(Evans et al. 2003;Georgeetal.2008; Gutzeit and Ludwig-
Müller 2014). Thus, a high work load is already required to
Tab l e 1 (continued)
Product Plant species Application Manufacturer Reference
Stem cell culture extracts: BerryFlux
Vita, Cell integrity, Bionymph
peptide, Cell Pulse, Daphne
VitaSense, FicuCell Vita, Hibiskin
Vita, Lykosin defense, Vita Freeze,
Mythus Vita, Vita Nova,
VitaShape, VitaLight
Rubus idaeus,Nicotiana sylvestris,
Psilanthus bengalensis,Daphne
odora,Opuntia ficus-indica,Hibiscus
syriacus,Solanum lycopersicum,
Actinidia deliciosa,Lotus japonicus,
Coleus forskohlii,Cirsium
eriophorum
Cosmetics: skin rejuvenation and
care, skin brightening and
firming
Vitalab
www.vitalabactive.com
Apone et al. (2010), Barbulova et
al. (2010), Bimonte et al.
(2011), Tito et al. (2011), Tito
et al. (2015), Di Martino et al.
(2017)
Plasma Rich in Cell Factors (PRCF)
products: Arabian Cotton,
Luminia Granatum, Sensia Carota
Gossypium herbaceum,Punica
granatum,Daucus carota sativa
Cosmetics: skin rejuvenation and
care, skin whitening
Vytrus Biotech
www.vytrus.com
Juanis (2017)
Phyto-Peptidic Fractions (PPF)
products: Capilia Longa
Curcuma longa Cosmetics: treatment of hair loss
PPF products: Centella Reversa Centella asiatica Cosmetics: skin rejuvenation and
care
1
No longer available
2
Now part of Symrise AG
3
Member of the Croda International Group
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Fig. 1 Schematic representation
of the procedure for the
establishment of a DDC-based
plant cell suspension culture
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identify the most promising candidates among the different
callus cell lines to be initiated, maintained, and mass
propagated. After the callus cell line selection, the
suspension cell culture is generated, as exemplarily
presented in Fig. 1. Calli are transferred from a petri dish
into a shake flask containing liquid culture medium. With
subsequent cultivation in a shaker, the size of the callus cell
aggregates becomes smaller. This improves mass transfer by
increasing the specific growth surface and implicates a higher
growth velocity of cells in the shake flask than in the petri
dish. A successive homogenization procedure as described
by Eibl et al. (2009a) reduces the time needed to provide a
homogeneously growing and producing plant suspension cul-
ture. Furthermore, it is important to mention that the culture
medium is often modified for maintenance, growth, and pro-
duction in terms of phytohormone type and concentration, and
levels of nitrogen, phosphate, and sucrose (Bhojwani and
Dantu 2013;Murthyetal.2014). This is done with
Murashige and Skoog (Murashige and Skoog 1962), Schenk
and Hildebrandt (Schenk and Hildebrandt 1972), or Gamborg
B5 medium (Gamborg et al. 1968), for example. DDC-
derived plant suspension cells reaching typical doubling times
of between 2 and 4 days normally grow in aggregates that
consist of up to hundreds of cells. The aggregate formation,
mainly ascribed to the formation of extracellular polysaccha-
rides in older cultures, may change the rheology of the culture
broth, limit mass transfer, and reduce cell growth and product
formation (Eibl et al. 2009a). Moreover, with increasing cul-
tivation time, genetic instabilities of the DDCs may occur,
caused by somaclonal variations and evidenced by decreased
or complete loss of product formation (Georgiev et al. 2009).
The availability of a working cell bank containing the cryo-
preserved, DDC-derived production suspension cell line re-
duces the risk of somaclonal variations because of reduced
subcultivation intervals. As in the case of mammalian suspen-
sion cultures, the controlled rate slow freezing approach is the
gold standard for DDC-based plant suspension cells
(Lawrence 2015;Schumacheretal.2015). However, in com-
parison to mammalian cells, cell regrowth is more difficult for
plant cells after thawing.
Due to the advantages of undifferentiated cambial meriste-
matic cells (CMCs) over DDCs, CMC-based plant suspension
cultures have gained increasing attention over the past few
years (Lee et al. 2010; Lee et al. 2012; Moon et al. 2015;
Ochoa-Villarreal et al. 2015;Sood2017). CMCs, which have
small spherical abundant vacuoles, are morphologically and
physiologically stable, grow as single cells, and are easy to
regrow after cryopreservation. Homogenization procedures
are becoming obsolete, and CMC-based suspension cultures
have a superior growth and production performance to that of
DDC-based ones. The company Unhwa, owner of the world’s
first patent for CMC isolation and cultivation, successfully
developed CMC-based suspension cultures of Taxus
cuspidata,Ginkgo biloba,andSolanum lycopersicum for ap-
plications in the cosmetics industry (Loake and Ochoa-
Villareal 2017).
Plant tissue cultures
For cosmetics and food products, plant tissue cultures play a
minor role in contrast to the previously described plant sus-
pension cultures. Thus, we give this topic only marginal con-
sideration. There are just a few product examples that are
based on hairy roots (e.g., Mibelle Biochemistry’s
RootBioTec HO from Ocimum basilicum) and somatic em-
bryo cultures (e.g., Vitalab’s Vita Nova from Lotus japonica).
Hairy roots (Fig. 2a) are generated following infection with
the soil bacterium Rhizobium rhizogenes (formerly
Agrobacterium rhizogenes), which shifts the transfer-DNA
(T-DNA) originating from the root inducing plasmid (Ri plas-
mid) into the plant genome. The successful transformation
process results in the formation of proliferating roots, so-
called hairy roots, on the explant infection side. Hairy root
cultures are characterized by lateral branching, similar growth
to plant suspension cultures, a hormone-free propagation pro-
cedure, a lack of geotropism, and genetic stability. But hairy
roots can only be applied to produce bioactive compounds
synthesized within the roots of the parent plant. Detailed in-
formation about hairy root culture establishment, mainte-
nance, and cultivation is provided by Georgiev et al. (2007),
Eibl et al. (2009a), Pistelli et al. (2011), Sharma et al. (2013),
and Sena (2015).
Somatic embryos delivering bioactivecompounds are mor-
phologically and physiologically identical to zygotic embryos
present in the seeds of the parent plant. They are induced by
either differentiated orundifferentiated somatic cells through a
series of morphological and biochemical changes. The devel-
opmental processes of the somatic embryos are regulated by
multiple factors, to which also phytohormones belong. For
more detailed information, the interested reader is referred to
Hess (1992), Quiroz-Figueroa et al. (2006), Tito et al. (2015),
and Jang et al. (2016). Figure 2b shows a somatic embryo
culture at the end of the somatic embryogenesis, known as
torpedo-stage embryos.
Plant cell culture propagation and extract
manufacture
Selection of the optimum cultivation parameters
and the most suitable bioreactor type
The plant cell culture type, in particular, its morphology,
growth, and production behavior, influences the selection of
the bioreactor type and the definition of its optimum cultiva-
tion parameters. By screening a highly productive production
8666 Appl Microbiol Biotechnol (2018) 102:8661–8675
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cell line, and optimizing the culture medium and environment,
up to 30-fold increases in product titers have been reported
(Ochoa-Villarreal et al. 2015).
A further stimulation of the secondary metabolism is
achievable by illumination with light (Curtin et al. 2003;
Cuperus et al. 2007; Tassoni et al. 2012) or/and elicitation
(Naik and Al-Khayri 2016;Sevón1997; Lijavetzky et al.
2008;Goeletal.2011;Zhouetal.2015). The agents used
for elicitation, the so-called elicitors, bind to specific receptors
on the outside of the cytomembrane of the plant cell and
trigger signaling cascades, which activate transcription of
genes for synthesis of phytoalexins, reactive oxygen com-
pounds, and defense enzymes. According to their origin, we
distinguish between biotic elicitors (e.g., cell wall and mem-
brane compounds, glycoproteins, modified nucleic acids) and
abiotic elicitors (e.g., ultraviolet radiation, heavy metals, heat,
cold). But establishing aneffective elicitation process requires
determination of the optimal elicitor type, dosage, and expo-
sure time and, thus, is very laborious. Nevertheless, elicitation
has been widely applied to increase the production of plant
cell culture-based secondary metabolites (Singh and Dwivedi
2018) and is regarded as most effective approach. As shown
by Jeandet et al., grape suspension cell culture-based produc-
tion of resveratrol is feasible in bioreactors with titers up to
7gL
−1
when the production process is induced by the com-
bination of methyl jasmonate and a cyclodextrin (Jeandet et al.
2016). This is the highest product titer reported in a plant cell
culture-based secondary metabolite production process so far.
Above all, elicitation may even cause the secretion of second-
ary metabolites, which typically are intracellular products.
Nowadays, the user can choose from numerous different
bioreactor types that have been used in cultivations with plant
cell cultures over decades. The selection of the bioreactor type
most suitable for a particular bioprocess is a very complex
task, as shown by Werner et al. for plant cell cultures. The
optimum bioreactor type should be well-instrumented as well
as scalable and should support the growth of the production
cell line and the formation of the desired bioactive com-
pound(s) while keeping the bioreactor footprint low. This
means that mixing gas supply and dispersion of the plant cell
culture have to be sufficient, while mass transfer limitations
and accumulations of harmful by-products should be avoided
(Werner et al. 2017). Excessively high shear forces in the
bioreactor, which result from high specific power input by
mixing and/or aeration, need to be excluded. Homogeneous
and sufficient illumination and dissipation of the heat for
photoauto- and photomixotroph plant cell cultures in the bio-
reactor are also required. However, demands in terms of the
tolerable oxygen transfer rate as well as power input and the
intensity (80.7–1345 μmol m
−2
s
−1
) and duration (0, 8, 16,
24 h) of illumination may differ for cell growth and product
formation (Chattopadhyay et al. 2002;Eibletal.2009a;
Hasan et al. 2017). Werner et al. (2017) propose selecting
the bioreactor type depending on the mostsuitable volumetric
oxygen transfer coefficient (k
L
a) to specific power input (P/V)
ratio in a first step. Subsequently, the design of the bioreactor
type (e.g., impeller type and number, sparger type) can be
modified if required and possible. Usage of computational
fluid dynamics (CFD) has been shown to be beneficial for
the optimization of bioreactor design and the definition of
the main process parameters (e.g., impeller speed, rocking
rate, rocking angel, aeration rate) to be realized. In principle,
it is possible to more rapidly develop and manufacture biore-
actor prototypes and to reduce the number of experiments by
applying CFD (Werner et al. 2014b).
Plant cell suspension lines, in particular those growing
slowly and behaving as Newtonian fluids, are very easy to
propagate in bioreactors. Attention must be paid to enabling
sufficient power input and oxygen when plant cell cultures
with non-Newtonian fluid flow behavior (usually fast growing
Fig. 2 Examples of plant tissue cultures. aHairy root culture of Ocimum
basilicum (the culture was established at the Technical University
Dresden, photo by Sibylle Kümmritz). bSomatic embryo culture of
Coffea canephora (the culture was established at the Nestlé R&D
Centre Tours as presented at the DECHEMA Himmelfahrtstagung 2018
in Magdeburg, Germany, poster contribution)
Appl Microbiol Biotechnol (2018) 102:8661–8675 8667
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plant cell suspension cultures) are to be proliferated without
damage by hydromechanical stress (Eibl et al. 2009b; Werner
et al. 2014a). As a general rule, plant cell suspension cells with
slow or moderate growth can be propagated in the same bio-
reactor types as mammalian suspension cells. The oxygen
demand is comparable, maximum oxygen uptake rates of be-
tween 2 and 10 mmol L
−1
h
−1
having been determined for
plant suspension cells (Curtis et al. 2006). But the majority
of the plant suspension cells tolerate higher hydromechanical
stress than mammalian suspension cells (Eibl et al. 2009a). An
even greater challenge than the cultivation of plant suspension
cells, characterized by a slow to moderate growth in bioreac-
tors, is that of fast growing plant suspension cells, hairy root
and somatic embryo cultures. It is a matter of fact that the
cultivation of fast growing suspension cell lines is often ac-
companied by strong foam formation and flotation. This does
not apply to hairy root and somatic embryo cultures, where
bioreactor types guaranteeing homogeneous power input, ox-
ygen, and light supply as well as avoiding high shear stress
peaks are preferred.
Most frequently used bioreactor types
Nowadays, stirred bioreactors, bubble column bioreactors, air-
lift bioreactors, and wave-mixed bioreactors with one-
dimensional (1-D) motion are most often usedfor commercial
productionswithplantcellcultures(Ruffonietal.2010;
Georgiev et al. 2013; Steingroewer et al. 2013; Stiles and
Liu 2013;Imsengetal.2014; Lehmann et al. 2014;Mamun
et al. 2015). The working principles of these four bioreactor
types are depicted in Fig. 3. When focusing on the cubic meter
scale, stainless steel stirred bioreactors (Fig. 3a) prevail. They
belong to mechanically driven bioreactors and are commonly
regarded as the system of choice for plant cell suspension
cultures. In contrast to their stirred counterparts, reusable bub-
ble column (Fig. 3b) and airlift bioreactors (Fig. 3c) have no
moving parts. They are pneumatically driven and are used to
mass propagate the more shear sensitive plant tissue cultures
(hairy root and somatic embryo cultures) up to cubic meter
scale. Bioreactor modifications, such as an increase in diame-
ter of the bioreactor head section to contribute to foam reduc-
tion, have been made (Paek et al. 2005; Jiang et al. 2015).
Such bubble column and airlift bioreactors with a balloon-
like head section are often referred to as Bballoon-type
systems.^
Where working volumes up to 300 L are sufficient, wave-
mixed bioreactors with 1-D motion (Fig. 3d) are frequently
operated with plant cell cultures. This bioreactor type belongs
to the group of so-called single-use bioreactors. The wave-
mixed bioreactor with 1-D motion has a multilayer plastic
bag as a cultivation container. The bag is provided ready-to-
use by the bioreactor supplier and is discarded after one single
usage. Lehmann et al. (2014) present single-use bioreactors
suitable for plant cell cultures in their review. Furthermore,
they have compiled bioengineering data for wave-mixed bio-
reactors with 1-D motion. These bioreactors are obtainable
from different suppliers at up to 600 L total volume and may
even be equipped with light-emitting diodes. By moving the
rocker unit, a wave is induced in the bag containing the culture
medium and cells. In this way, mixing occurs and surface aer-
ation takes place while the medium surface is permanently
renewed. The power input is controllable by the rocking angle,
Fig. 3 Schematic diagrams of instrumented bioreactors preferred in
commercial production processes with plant cell cultures which
generate products for the cosmetics and food industries. aStirred
bioreactor. bBubble column bioreactor. cAirlift bioreactor. dWave-
mixed bioreactor with 1-D motion
8668 Appl Microbiol Biotechnol (2018) 102:8661–8675
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rocking rate, and filling level of the bag. Wave-mixed bioreac-
tors with 1-D motion are suitable for both plant cell suspension
cells and tissue cultures. Normally, an antifoam agent is not
required, because the foam is constantly incorporated into the
culture broth. At high cell densities and culture broth viscosi-
ties, mass transfer can be limited in wave-mixed bioreactors
with 1-D motion. A solution might be the application of a
wave-mixed bioreactor with multi-dimensional motion such
as the Cell tainer (Oosterhuis and Junne 2016). However, no
reports about its usage forcommercial cultivations of plant cell
cultures have appeared in the literature to date.
In addition, there are a high number of further suitable
bioreactor types for plant cell cultures. However, they are
partially non-instrumented and have been used only for re-
search purposes or displayed in-house developments of re-
search groups and companies. Examples are temporary im-
mersion systems (Ducos et al. 2007;Ducosetal.2010;
Georgiev et al. 2014), rotating drum bioreactors (Tanaka et
al. 1983;Georgievetal.2013), mist bioreactors (Weathers et
al. 2008; Weathers et al. 2010; Fei and Weathers 2014), and
bioreactor systems with orbitally shaken bags (Werner et al.
2013; Lehmann et al. 2014).
Plant cell culture extract manufacture
The process of extract manufacture following cultivation in a
bioreactor ishighly dependent on the chemical nature(s) of the
bioactive substance(s) to be contained in the plant cell culture
extract. Moreover, whether the extract is a liquid or a powder
needs to be taken into account. A distinction is made between
hydrosoluble (e.g., amino acids, glucides, flavonoids, antho-
cyanins, phenolic acids) and liposoluble (e.g., vitamins, to-
copherols, fatty acids) compounds and those derived from
plant cell walls (mixtures of peptides and sugars).
Independent of the extract type, the first step is always to
harvest the culture broth containing the intracellular target
compound(s). The subsequent operations are manufacturer
specific and generally include harvest, homogenization and
disruption of the cell mass, extraction with solvents or proteo-
lytic enzymes and/or chromatographic methods, and washing
steps (Venkatramesh et al. 2010; Barbulova et al. 2014;Morus
et al. 2014). Furthermore, if the extract is a powder, a drying
process with freeze dryers, spray dryers, or vacuum dryers is
required. There are also manufacturing processes for plant cell
culture extracts that differ from the above, the details of which
can be found in the manufacturers’patent documents. One
example is Mibelle Biochemistry’s process for extract manu-
facture of the PhytoCELLTECH actives (Table 1). After a
mixing process with liposomes, phenoxyethanol, and antiox-
idants (ascorbic acid or tocopherol), a liquid extract is pro-
duced from all the compounds, including plant cells that have
been disrupted by high-pressure homogenization at 1500 bar
(Blum et al. 2013).
It is important that plant cell culture extracts for the cos-
metics and food industries are not of toxicological concern
when it comes to their final use. In the case of supplement
ingredients contained in very low concentrations in the final
product, a risk scenario for the consumer is rather unlikely. In
cosmetics products, for example, the levels are often below
1%. The replacement of traditional, synthetic phytohormones
in the culture medium (2.4-dichlorophenoxyacetic acid, 6-
bezylaminopurine, N6-furfuryladenine) with natural phyto-
hormones (indole-3-acetic acid, zeatin) and the application
of phytohormone elicitors (jasmonic acid, methyl jasmonate,
salicylic acid) and/or light further increase the safety of plant
cell culture-based products for the cosmetics and food indus-
tries (Murthy et al. 2015). However, more attention needs to
be paid to plant cell culture-based food supplements and food.
When the plant cell culture is the foodstuff itself, not only does
ariskanalysishavetobecarriedoutbyatoxicologistbuta
food-conform culture medium is also necessary.
Latest developments in plant cell
culture-based food production
Example 1: cell culture chocolate
The megatrends of society, health, individualization, and mo-
bility have effects on consumer behavior in terms of food
culture and resulting developments in the food industry
(Berghofer et al. 2015; Reynolds 2016). According to the
recent European Food Trends Report of the Gottlieb
Duttweiler Institute, there are two main trends, a healthy
way of eating and high tech food (Schäfer et al. 2017).
Customers want to be actively involved in the production,
distribution and consumption of their food. Plant cell cultures
provide innovative solutions in this context. As mentioned
above, their establishment is independent of location, and their
metabolism can effectively be influenced (e.g., by medium
composition and/or cultivation parameters) during the mass
propagation procedure. On the one hand, it is possible to pro-
duce bioactive compounds which are responsible for food
aroma and taste and/or have health benefits. On the other
hand, the formation of harmful compounds can be reduced
or completely suppressed by working with plant cell cultures.
Such an approach has been implemented in a Zurich
University of Applied Sciences (ZHAW) study which inves-
tigated the potential of callus and suspension cell lines of
Theobroma cacao for the cocoa ingredient in chocolate pro-
duction. In contrast to the developments published by Diana
Plant Sciences (Venkatramesh et al. 2010;Barney2013),
where the cell cultures originated from immature T. cacao
floral explants, cocoa beans (seeds; Fig. 4a) of cocoa fruits
(origin: USDA-ARS Tropical Agriculture Research Station
in Puerto Rico, 2200 P.A. Campos Ave., Suite 201,
Appl Microbiol Biotechnol (2018) 102:8661–8675 8669
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Mayaguez, Puerto Rico) characterized by different stages of
maturity were used in our lab (Stutz 2018). Four callus cell
lines grown at 29 °C on a modified Murashige and Skoog
medium in the dark reached doubling times of 7 days.
Whereas these callus cultures had comparable or even up to
40% higher polyphenol content (epicatechin, procyanidins
B1, B2, C1, and cinnamtannin A2) than the source material
(T. cacao beans), the alkaloids caffeine and theobromine were
reduced by up to 100%. Furthermore, an overexpression of the
amino acids valine, cysteine, and phenylalanine, which are
known to boost the immune system, was detected. Because
the callus clone ICS-45 (Fig. 4b) was also very friable, it was
decided to generate the production suspension cell line from it
(Fig. 4c). The T. cacao suspension cells reached doubling
times of 4 days when propagated in 1-L shake flasks over
9 days in batch mode (300 mL working volume, 29 °C,
120 rpm, 25 mm shaker amplitude). The single cells of T.
cacao had a spherical shape and a diameter between 25 and
50 μm, which is about 50% smaller than usual. With increas-
ing cultivation time, the T. cacao suspension cells propagated
in a modified Murashige and Skoog medium tended to grow
in large clusters, the vast majority of which appeared slightly
brownish under the microscope.
Based on the results of the shake flask runs, the process was
transferred to a wave-mixed single use bioreactor in order to
generate sufficient T. cacao cell mass for the production of cell
culture chocolate. We worked with Sartorius Stedim’s
BIOSTAT RM 20/50 equipped with a 20-L bag with screw
cap (Fig. 4d). A feed with 5-L medium was realized on day 7
(initial working volume 6 L). About 300 g biomass (fresh
weight) was harvested on day 16, separated from the culture
broth, rinsed, and freeze-dried (Fig. 4e). The in vitro cocoa
powder was used to produce three bars of 70% dark chocolate
(Fig. 4f). While the biomass was not pre-treated for the first
bar, the biomass of the second bar was completely aerobically
incubated (46 h) and that for the third bar both anaerobically
(30 h) and aerobically (16 h) incubated. The purpose of this
procedure was to simulate the fermentation of the cocoa
beans, which is crucial for the development of aroma in the
traditional production of chocolate. The cocoa powder was
roasted, sugar and cocoa butter were added, and the mixture
was rolled. Before lecithin was added, the chocolate mass was
heated up. Finally, the chocolate mass was casted into forms.
Professional chocolate tasting was carried out by a ZHAW
expert panel, who confirmed that the cell culture chocolate
provided a unique taste experience. Interestingly, the untreated
bar of chocolate performed best. An intense and complex
aroma was described, whereby citrus and berry aromas were
predominant. Beyond that, lactic, malty and green tones, and a
mildly acidic component were detected in the profile. The
different expressions were also confirmed by a first aroma
analysis (volatile aroma compounds). However, our investi-
gations are still ongoing, and future studies will include, for
example, the increase in process efficiency.
Fig. 4 Main steps of the production of chocolate based on Theobroma
cacao suspension cells. aOne of the cacao fruits used to induce seven
callus culture cell lines from beans. bEstablished callus culture of the
clone ICS-45. cMicroscopic picture of T. cacao suspension cells growing
in shake flasks (clone ICS-45). dTwenty-L Flexsafe bag with mass
propagated T. cacao suspension cells. eT. cacao suspension cells after
freeze drying. fProduced cell culture chocolate
8670 Appl Microbiol Biotechnol (2018) 102:8661–8675
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Example 2: plant cell culture-based foodstuff
produced in a home bioreactor
Another interesting approach for modern plant cell culture-
based food production is used by specialists from the
Technical Research Center of Finland Ltd. (VTT), who devel-
oped a bioreactor to produce about 500 g (fresh weight) of
edible plant cell culture biomass within 1 week at home. The
bioreactor, referred to as BHome bioreactor^and working in a
similar way to a coffee machine, is shown in Fig. 5. Its current
design is about the size of a table lamp and consists of a
container with a lid, which has been manufactured with a
three-dimensional printer. The bioreactor container has two
openings: one for the insertion of a single-use bag or capsule
with the cell starter (plant cell culture with medium) and one
for adding water. By turning on the bioreactor, whose temper-
ature is controlled, and which can be illuminated and aerated,
the cell culture is kept at optimal growth conditions (Räty
2017). Although this bioreactor, which was listed among the
ten Forbes’s food trends of 2017 (Lempert 2016), has already
been successfully applied to mass propagate suspension cul-
tures of blackberries and cloudberries in first tests, it is not yet
ready for the market. Moreover, additional questions
concerning sterility, cell culture inoculum (stable, easy to han-
dle, full-bodied taste), and culture medium (food-conform,
cheap) have to be addressed.
All in all, it is expected that this bioreactor, including cell
cultures and appropriate culture media, will be available on
the market within the next 9 years. The idea of cooking with
plant cell cultures that have been mass propagated in con-
sumers’own kitchens should then become a reality.
Conclusions
The use of plant cell cultures instead of whole plants allows
products for the cosmetics and food industries to be
manufactured with less energy, lower possible impacts on
the environment, and independent of location and season.
The currently available products are supplement ingredients
to reduce hair loss and aging of skin, which improve skin
quality and strengthen the body’s immune defense system.
In today’s commercialmanufacture, plant cell suspension cul-
tures are grown in reusable stainless steel stirred bioreactors or
single-use wave-mixed bioreactors with 1D-motion for the
most part. Due to small working volumes, higher safety, more
rapid and simple putting into operation, and shorter develop-
ment times, single-use wave-mixed bioreactors are ideal for
research and development and for personalized products.
However, with very few exceptions, these bioreactors were
originally designed for pharmaceutical high-value products
based on mammalian cell cultures. The demand for bioreac-
tors providing bioactive substances for the pharmaceutical
industry is higher than in the cosmetics and food industries,
where the lower demand for plant cell culture-based produc-
tions of cosmetics and food supplement ingredients means
that they are often too expensive.
In line with the current trend for home-made food, there is a
need for low-cost bioreactors which are easy to handle and
provide cellmass in the three-digit and four-digit g-range. The
Finnish BHome bioreactor^is a first approach but has not yet
been commercialized and made suitable for everyday use. In
addition to this novel bioreactor, it should be ensured that the
user has access to inoculum cultures and suitable food-
compatible culture media in the future. Indeed, it seems that
the way to a food revolution has been paved and plant cell
culture-based superfood independent of location will soon be
obtainable.
Acknowledgements A big thank you goes to Ansgar Schlüter, Carlo
Weber, Vasilisa Pedan, Karin Chatelain, and Petra Huber, who made the
practical production ofcell culture chocolate in the ZHAW labs possible.
Funding information The School N at the Zurich University of Applied
Sciences (ZHAW) financially supported our feasibility study on produc-
ing a cell culture chocolate.
Fig. 5 VTT’s home bioreactor, which was developed in co-operation
with designers at the Aaolto University School of Arts, Design and
Architecture, and which may be applied to produce berry cell culture
biomass for the morning-cereal or smoothie, or which can be eaten as
supplement in the future (with kind permission of Dr. Heiko Rischer,
VTT Technical Research Centre of Finland Ltd.)
Appl Microbiol Biotechnol (2018) 102:8661–8675 8671
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with animals
performed by one of the authors.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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