Name: employing aeroponic systems for the clonal propagation of cannabis
Uploaded: 2021-12-01T13:00:00
Description: Watch this Scientific Journal Video about Employing Aeroponic Systems for the Clonal Propagation of Cannabis at JoVE.com

This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo and the Ministry of Science and ICT (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021), The authors also wish to thank Justin Henderson at Summit CBD for the generous donation for &#34;Cherry Wine&#34; seeds.

1. Generation of a mother plant for clonal propagation
<ol>
	<li>Select a healthy, female mother plant that exhibits desirable morphological and chemical characteristics specific to its intended use.</li>
	<li>Allow the mother plant to reach the appropriate size (roughly 25 mature shoots) for clonal propagation (i.e., cuttings).</li>
	<li>Allow the mother plants to remain in the vegetative growth stage (light: dark = 18 h:6 h) to promote shoot growth for future propagation.</li>
</ol>
<p class="jove_ti...

<ol>
	<li>ElSohly, M. A., Radwan, M. M., Gul, W., Chandra, S., Galal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemistry+of+Cannabis+sativa+L.">Phytochemistry of Cannabis sativa L.</a> Progress in the Chemistry of Organic Natural Products. 103, 1-36 (2017).</li><li>Cunetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+pain+treatment+with+cannabidiol+in+kidney+transplant+patients+in+Uruguay.">Chronic pain treatment with cannabidiol in kidney transplant patients in Uruguay.</a> Transplantation Proceedings. 50 (2), 461-464 (2018).</li><li>Hausman-Kedem, M., Menascu, S., Kramer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+CBD-enriched+medical+cannabis+for+treatment+of+refractory+epilepsy+in+children+and+adolescents+-+An+observational,+longitudinal+study.">Efficacy of CBD-enriched medical cannabis for treatment of refractory epilepsy in children and adolescents - An observational, longitudinal study.</a> Brain &amp; Development. 40 (7), 544-551 (2018).</li><li>Linge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+induces+rapid-acting+antidepressant-like+effects+and+enhances+cortical+5-HT/glutamate+neurotransmission:+role+of+5-HT1A+receptors.">Cannabidiol induces rapid-acting antidepressant-like effects and enhances cortical 5-HT/glutamate neurotransmission: role of 5-HT1A receptors.</a> Neuropharmacology. 103, 16-26 (2016).</li><li>Lehmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cannabidiol+treatment+reduces+early+pancreatic+inflammation+in+type+1+diabetes.">Experimental cannabidiol treatment reduces early pancreatic inflammation in type 1 diabetes.</a> Clinical Hemorheology and Microcirculation. 64 (4), 655-662 (2016).</li><li>Shannon, S., Lewis, N., Lee, H., Hughes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+in+anxiety+and+sleep:+A+large+case+series.">Cannabidiol in anxiety and sleep: A large case series.</a> The Permanente Journal. 23, 18-41 (2019).</li><li>Russo, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+cannabis+and+its+preparations+in+saga,+science,+and+sobriquet.">History of cannabis and its preparations in saga, science, and sobriquet.</a> Chemistry &amp; Biodiversity. 4 (8), 1614-1648 (2007).</li><li>Vera, C. L., Hanks, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemp+production+in+Western+Canada.">Hemp production in Western Canada.</a> Journal of Industrial Hemp. 9 (2), 79-86 (2004).</li><li>. Hot hemp: How high THC levels can ruin a legal hemp harvest Available from: <a target="_blank" href="https://www.westword.com/marijuana/hot-hemp-how-high-thc-levels-can-ruin-a-legal-hemp-harvest-9963683">https://www.westword.com/marijuana/hot-hemp-how-high-thc-levels-can-ruin-a-legal-hemp-harvest-9963683</a> (2018)</li><li>Lata, H., Chandra, S., Techen, N., Khan, I. A., ElSohly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+genetic+stability+of+micropropagated+plants+of+Cannabis+sativa+by+ISSR+markers.">Assessment of the genetic stability of micropropagated plants of Cannabis sativa by ISSR markers.</a> Planta Medica. 76 (1), 97-100 (2010).</li><li>Caplan, D., Dixon, M., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+rate+of+organic+fertilizer+during+the+flowering+stage+for+Cannabis+grown+in+two+coir-based+substrates.">Optimal rate of organic fertilizer during the flowering stage for Cannabis grown in two coir-based substrates.</a> HortScience. 52 (12), 1796 (2017).</li><li>Monthony, A. S., Page, S. R., Hesami, M., Jones, A. M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+of+Cannabis+sativa+tissue+culture.">The past, present and future of Cannabis sativa tissue culture.</a> Plants (Basel). 10 (1), 185 (2021).</li><li>Clarke, R. C., Merlin, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabis+domestication,+breeding+history,+present-day+genetic+diversity,+and+future+prospects.">Cannabis domestication, breeding history, present-day genetic diversity, and future prospects.</a> Critical Reviews in Plant Sciences. 35 (5-6), 293-327 (2016).</li></ol>

With the increasing demand for Cannabis plants with consistent cannabinoid content, various clonal propagation methods have been exploited in Cannabis industry. The asexual propagation shows several advantages over sexual methods for large-scale, consistent production. An aeroponic propagation system is a modified version of a hydroponic system that utilizes an aerated nutrient-rich water mist to provide rapid root development. The described aeroponic system is composed of three critical steps, 1) gener...

Cannabis sativa L. is an annual, dioecious, flowering plant classified in the family Cannabaceae. Cannabinoids, produced predominantly within glandular trichomes located on the outer epidermal layer of bract tissues on female inflorescences1, are becoming an increasingly popular research topic, primarily due to their progressively recognized medicinal properties. Cannabidiol (CBD) is the second most prominent cannabinoid found in Cannabis after &#916;9-tetrahydrocannabinol (THC) and is attributed to a host of medicinal benefits, including analgesic properties2, anti-seizure properties3, antidepressant properties4, reducing the risk of diabetes5, and treating various sleep disorders6. Due to the multitude of health benefits associated with the metabolites of the Cannabis plant, there is a growing demand for its commercial-scale production7. To meet this demand, cultivation methods are constantly being improved and reinvented to continuously supply consistent, high-quality plant material to the emerging Cannabis industry.
The propagation of Cannabis can be facilitated in two ways: sexual or asexual reproduction. An example of sexual reproduction is pollinating a female ovule with pollen from a male&#39;s stamen resulting in a seed that can be germinated. Seed germination is a reliable cultivation method that has been used for breeding and cultivation purposes where desirable phenotypical traits are selected in parental lines to improve the quality of the offspring Cannabis plants, including traits such as drought tolerance, insect resistance, increased yield, and increased potency8. However, unintended cross-pollination is an inherent risk when performing sexual reproduction, causing undesirable offspring, which leads to the potential loss of desirable traits or an introduction of unwanted traits. An example of this unintended pollination is highlighted by hemp growers receiving hemp seed pollinated with THC- producing pollen resulting in significant economic loss due to the non-compliant plants (&#62;0.3% total THC w/w)9. Additionally, to generate a crop that consists of only females, a feminized seed must be sown instead of a non-feminized seed, which can lead to hermaphroditism and other undesirable traits leading to economic loss. To overcome the limitation of sexual reproduction of Cannabis, asexual reproduction has been widely practiced in commercial production models of the Cannabis industry10.
Asexual reproduction of Cannabis requires only a single plant, which allows for the multiplication of a single genotype that allows for commercial production of plants carrying desirable agronomic and pharmaceutical traits. A common form of asexual Cannabis reproduction is to cut and insert small portions of a female plant into a soilless substrate11 which is covered by a humidity dome to induce root formation. Although this method has proven successful, a common drawback is the accumulation of a high level of humidity (usually 80% or higher) inside the dome, providing an ideal growth environment for fungal pathogens, which can be detrimental to new, sensitive cuttings. Another form of asexual propagation is micropropagation using tissue culture, where sterile techniques allow for the propagation of insect, microbe, and virus-free Cannabis plant material in limited space12. This process, however, is expensive, time-consuming and requires trained laboratory technicians which are generally inaccessible for large-scale Cannabis facilities.
Very few published research reports exist on the clonal propagation of Cannabis. In order to provide a basis for the understanding of asexual reproduction of Cannabis for research purposes and industrial production, this study aimed to demonstrate the ease and accessibility of employing aeroponic systems for the clonal propagation of Cannabis. Aeroponic systems are ideal for the asexual propagation of Cannabis, consistently supplying nutrient-rich water to the cuttings, inducing early root formation in a timely manner, and allowing for a plant to be maintained indefinitely if needed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-part Fox Farm</td>
			<td>Fox Farm</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Promix</td>
			<td>Promix</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Roots Organic Original</td>
			<td>Auora Innovations</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Wiggle Worm Earth Worm Castings</td>
			<td>UNCO Industries</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>Algae and Bacterial Cleaning Solution (Clear Rez)</td>
			<td>EZ Clone</td>
			<td>SKU#: 225</td>
			<td>8 fl. Oz.</td>
		</tr>
		<tr>
			<td>Artificial Lighting</td>
			<td>AgroBrite</td>
			<td>SKU#: 1399</td>
			<td>T5 324W 4&#39; 6-Tube Fixture with Lamps</td>
		</tr>
		<tr>
			<td>Cannabis Mother plant 1 (Cherry Wine)</td>
			<td>Summit CBD</td>
			<td>N/A</td>
			<td>Donated material</td>
		</tr>
		<tr>
			<td>Cannabis Mother Plant 2 (Red Wine)</td>
			<td>Trilogene</td>
			<td>SKU: 0101RR</td>
			<td></td>
		</tr>
		<tr>
			<td>Corresponding Plastic Lid</td>
			<td>Office Depot</td>
			<td>N/A</td>
			<td>38.1 cm x 25.4 cm</td>
		</tr>
		<tr>
			<td>Drill Bit 1</td>
			<td>Dewalt</td>
			<td>DW1586</td>
			<td>38.1 mm spade drill bit</td>
		</tr>
		<tr>
			<td>Drill Bit 2</td>
			<td>Dewalt</td>
			<td>DW1308</td>
			<td>3.175 mm drill bit</td>
		</tr>
		<tr>
			<td>Flora/Bloom (Nutrient Solution)-5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 726</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) (Available Phosphate 5.0%, Soluble Potash 4.0%, Magnesium 1.5%, Sulfur 1.0%)</td>
		</tr>
		<tr>
			<td>FloraGrow (Nutrient Solution)- 5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 724</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) ((Total Nitrogen 2.0% (0.25% Ammoniacal Nitrogen, 1.75% Nitrate Nitrogen), Available Phosphate 1.0%, Soluble Potash 6.0%, Magnesium 0.5%))</td>
		</tr>
		<tr>
			<td>FloraMicro (Nutrient Solution)- 5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 759</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) ((Total Nitrogen 5.0% (0.3% Ammoniacal Nitrogen, 4.7% Nitrate Nitrogen), Soluble Potash 1.0%, Calcium 5.0%, Boron 0.01%, Cobalt 0.0005%, Copper 0.01%, Iron 0.1%, Manganese 0.05%, Molybdenum 0.0008%, Zinc 0.015%))</td>
		</tr>
		<tr>
			<td>Horticultural Scissors</td>
			<td>Shear Perfection</td>
			<td>SKU#: 12620</td>
			<td>Platinum Stainless Steel Bonsai Scissors (2.4&#34;)</td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Equate</td>
			<td>Walmart # 574133562</td>
			<td>70% concentration</td>
		</tr>
		<tr>
			<td>Nutrient Mist Solution (Clonex Mist)</td>
			<td>Growth Technology</td>
			<td>SKU#: 4889</td>
			<td>10.14 fl. Oz (300 ml) (Total Nitrogen: 5.9 &#215; 10-4 %, Available Phosphate: 4.0 &#215; 10-4 %, Soluble Potash: 5.0 &#215; 10-4 %)</td>
		</tr>
		<tr>
			<td>pH Down</td>
			<td>General Hydroponics</td>
			<td>SKU#: 733</td>
			<td>946 ml (1 Quart) 2.43 lbs. (1.1 kg)</td>
		</tr>
		<tr>
			<td>pH Up</td>
			<td>General Hydroponics</td>
			<td>SKU#: 730</td>
			<td>946 ml (1 Quart) 2.43 lbs. (1.1 kg)</td>
		</tr>
		<tr>
			<td>Plastic Container</td>
			<td>Office Depot</td>
			<td>N/A</td>
			<td>38.1 cm x 25.4 cm x 30.48 cm</td>
		</tr>
		<tr>
			<td>Power Drill</td>
			<td>Dewalt</td>
			<td>DCD709B</td>
			<td>20-Volt Max &#189;&#8221; Drill</td>
		</tr>
		<tr>
			<td>Rockwool Cubes</td>
			<td>Grodan</td>
			<td>SKU#: 830</td>
			<td>38.1 mm</td>
		</tr>
		<tr>
			<td>Rooting Solution (Clonex Rooting Gel)</td>
			<td>Growth Technology</td>
			<td>SKU#: 939</td>
			<td>3.4 fl. Oz. (100 ml) (Indolebutyric Acid - 0.31%)</td>
		</tr>
		<tr>
			<td>Statistic Software (Prism)</td>
			<td>GraphPad Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submersible Water Pump</td>
			<td>ActiveAQUA</td>
			<td>SKU: AAPW250</td>
			<td>Model: AAPW250, Voltage 120V, Power 16W</td>
		</tr>
	</tbody>
</table>

employing aeroponic systems for the clonal propagation of cannabis

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings were excised from two hemp varieties, named &#34;Cherry Wine&#34; and &#34;Red Robin&#34; (17-20% w/w CBD), that served as &#39;mother plant&#39;. An auxin precursor (indole-3-butyric acid) was applied to stimulate root development in the basal portion of the excised cuttings prior to placement in the system. Cuttings were lightly misted with the nutrient mist solution every three days to provide nutritional support as the solution contains the essential macronutrients, including nitrogen, phosphorus, and potassium. The aeroponic system water reservoir maintained a pH range between 5.0-6.0 and a water temperature between 20-22 &#176;C. A submersible water pump was used to deliver water to the cuttings. The shoot tip cuttings were provided with 24 h of light per day for 10 days until root development occurred, upon which the rooted cuttings were transplanted for research purposes. These aeroponic systems have proven to generate desirable results for Cannabis propagation. The method described here alleviates potential time constraints that arise from traditional methods to allow for a more efficient means for the asexual propagation of Cannabis.

To validate the efficiency of the described aeroponic system, a total of 10 and 12 healthy 14 cm long shoots were excised from the mother plants, &#39;Cherry Wine&#39; and &#39;Red Robin&#39;, respectively (Figure 1A,B). After dipping into rooting induction media, the clones were placed into the system (Figure 2A). The construction and operation of an aeroponic system is shown as a schematic diagram in Figure 2A.</p...

Watch this Scientific Journal Video about Employing Aeroponic Systems for the Clonal Propagation of Cannabis at JoVE.com

Employing Aeroponic Systems for the Clonal Propagation of Cannabis

This protocol is designed to provide instructional information for the clonal propagation of Cannabis sativa L. by implementing aeroponic systems. The method described here includes all necessary supplies and protocols to successfully reproduce desirable morphological and chemical properties in the genus Cannabis.

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings ...

This protocol can aid in making an informed decision as to how to proceed with the regeneration of desired cannabis genetics in research or commercial production settings. The main advantage of this technique is inducing early root formation in cannabis cuttings while limiting their susceptibility to potential pathogens. The implications of this technique extend toward the improvement of challenges observed in cannabis propagation by inducing early root formation in cuttings, which can be applied in research or production settings.With a sterilized scalpel or scissor, cut the shoots with several nodes of approximately 10 centimeters in length near the apical meristem at an angle 45 degrees. Remove all foliage except that present on the top three nodes. Dip the newly excised cutting at approximately two to five centimeters up from the base of the stem into the rooting solution containing indole-3-butyric acid for approximately five seconds.Insert the cutting into the center of a rockwool cube til the depth of approximately two centimeters above the cube's base, positioned in the aeroponic system. Spray the unrooted cuttings with a nutrient mist solution every three days. Grow cuttings for about 18 to 24 hours in light per day with a photosynthetic photon flux density of 100 micromoles per second per square meter.Every two to five days, replenish the system with water at a pH between five to six. Lightly mist the cuttings with nutrient mist solution every three days and pour five milliliters of nutrient solution into the reservoir every three to five days. Add 15 milliliters of the algae and bacteria cleaning solution, containing 0.028%hypochlorous acid every five days.Select the cuttings with long, white fibrous roots. Carefully dislodge the rockwool cube from the system and untangle the roots. Transplant the cannabis propagules to a four liter nursery pot filled with a nutritious soil mix.The figure shows clones that developed roots in three to seven days and fully developed roots 37 centimeters in length after 10 to 14 days on the system, planted into a soil-filled pot. The average length of shoots and roots were approximately 24.8 centimeters and 37.8 centimeters for Cherry Wine and approximately 21.4 centimeters and 39.7 centimeters approximately for Red Robin, respectively. The differences between the two varieties were analyzed by two-way ANOVA followed by Tukey's multiple compassions test, showing no significant differences in shoot and root lengths between the two varieties.Selecting healthy cuttings exhibiting vigorous new growth is ideal for propagation. Maintaining the water level, temperature, pH, and nutritional aspects of the system reservoir are key for fast and effective outcomes.

employing-aeroponic-systems-for-the-clonal-propagation-of-cannabis

Research

JoVE Journal

Bioengineering

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...